Age-related macular degeneration (AMD) is a major cause of blindness in the developed world. Oxidative stress and inflammation are implicated in AMD, but precise mechanisms remain poorly defined. Carboxyethylpyrrole (CEP) is an AMD-associated lipid peroxidation product. We previously demonstrated that mice immunized with CEP-modified albumin developed AMD-like degenerative changes in the outer retina. Here, we examined the kinetics of lesion development in immunized mice and the presence of macrophages within the interphotoreceptor matrix (IPM), between the retinal pigment epithelium and photoreceptor outer segments. We observed a significant and time-dependent increase in the number of macrophages in immunized mice relative to young age-matched controls prior to overt pathology. These changes were more pronounced in BALB/c mice than in C57BL/6 mice. Importantly, IPM-infiltrating macrophages were polarized toward the M1 phenotype but only in immunized mice. Moreover, when Ccr2-deficient mice were immunized, macrophages were not present in the IPM and no retinal lesions were observed, suggesting a deleterious role for these cells in our model. This work provides mechanistic evidence linking immune responses against oxidative damage with the presence of proinflammatory macrophages at sites of future AMD and experimentally demonstrates that manipulating immunity may be a target for modulating the development of AMD.

Age-related macular degeneration (AMD) is a major disease affecting central vision, but the pathogenic mechanisms are not fully understood. Using a mouse model, we examined the relationship of two factors implicated in AMD development: oxidative stress and the immune system. Carboxyethylpyrrole (CEP) is a lipid peroxidation product associated with AMD in humans and AMD-like pathology in mice. Previously, we demonstrated that CEP immunization leads to retinal infiltration of pro-inflammatory M1 macrophages before overt retinal degeneration. Here, we provide direct and indirect mechanisms for the effect of CEP on macrophages, and show for the first time that antigen-specific T cells play a leading role in AMD pathogenesis. In vitro, CEP directly induced M1 macrophage polarization and production of M1-related factors by retinal pigment epithelial (RPE) cells. In vivo, CEP eye injections in mice induced acute pro-inflammatory gene expression in the retina and human AMD eyes showed distinctively diffuse CEP immunolabeling within RPE cells. Importantly, interferon-gamma (IFN-γ) and interleukin-17 (IL-17)-producing CEP-specific T cells were identified ex vivo after CEP immunization and promoted M1 polarization in co-culture experiments. Finally, T cell immunosuppressive therapy inhibited CEP-mediated pathology. These data indicate that T cells and M1 macrophages activated by oxidative damage cooperate in AMD pathogenesis.

Elevated levels of lysophosphatidylcholine (lysoPC), present in oxidatively damaged low-density lipoprotein (oxLDL), are implicated in cardiovascular complications. LysoPC is generated by free radical-catalyzed oxidation of polyunsaturated PCs to oxidatively-truncated phosphophatidylcholines (oxPCs). It is known that oxPCs are especially susceptible to hydrolysis by platelet-activating factor acetylhydrolase, a phospholipase (PL) A 2 that exists in plasma largely in association with LDL. Drugs that aim to prevent the generation of lysoPC by inhibiting this PLA 2 -catalyzed hydrolysis are in advanced clinical trials. We now report that spontaneous deacylation oxPCs, such as 1-palmityl-2-(4-hydroxy-7-oxo-5-heptenoyl)-sn-glycero-3-phosphocholine, occurs readily under physiological conditions of temperature and pH (t 1/2 = 30 min at 37 °C and pH 7.4). We also show that this reaction proceeds through an intramolecular transesterification mechanism. Because anti-phospholipase drugs cannot block this nonenzymatic pathway to lysoPC, additional therapeutic measures may be needed to avoid the pathological consequences of the newly discovered biomolecular chemistry of oxPCs.

We report that protein adducts of iso [4]levuglandin E 2 (iso [4]LGE 2 ), a highly reactive product of free radical-induced lipid oxidation, accumulate in human glaucomatous trabecular meshwork (TM) but not in controls. Reactive oxygen species play a pathogenic role in primary open angle glaucoma by fostering changes that reduce permeability of the TM tissue and consequently impede aqueous humor outflow resulting in elevated intraocular pressure. IsoLGs covalently modify proteins and are especially effective in causing protein-protein crosslinking. We found elevated levels of calpain-1 in glaucomatous TM. However, calpain activity in glaucomatous TM is only about 50% to that in controls. This paradox is explicable by the fact that modification by isoLGs renders calpain-1 inactive. Thus, treatment of calpain-1 with iso [4]LGE 2 in vitro results in covalent modification, inactivation, the formation of high molecular weight aggregates (as determined by Western and dynamic light scattering analyses), and resistance to proteasomal digestion. Iso [4]LGE 2 -modified calpain-1 undergoes ubiquitination and its loading impairs the cellular proteasome activity consistent with competitive inhibition and formation of suicidal high molecular weight aggregates. These data suggests that interference with proteasomal activity, owing to protein modification by isoLGs could contribute to glaucoma pathophysiology by decreasing the ability of the TM to modulate outflow resistance. KeywordsGlaucoma; Intraocular pressure; isolevuglandin; Primary open angle glaucoma; Retinal ganglion cell; Reactive oxygen species; Trabecular meshwork Glaucomas are a group of irreversible blinding neurodegenerative diseases that are late onset and progressive in nature. They are designated primary, when they occur without a known cause, or secondary, when onset can be attributed to any other illness or injury. World wide about 70 million people suffer from glaucoma 1 . Primary open angle glaucoma (POAG) is characterized by changes that reduce permeability of the trabecular meshwork (TM) tissue and consequently impede aqueous humor outflow resulting in elevated intraocular pressure (IOP). Mounting evidence supports the hypothesis that reactive oxygen species (ROS) play a pathogenic role in POAG 2 . Elevated levels of hydrogen peroxide in the aqueous humor promote TM degeneration and outflow resistance. Protection against the deleterious effects of ROS should be provided by the antioxidant activities of superoxide dismutase and glutathione Address Correspondence to: Sanjoy K. Bhattacharya, Bascom Palmer Eye, Institute, 1638 NW 10 th Avenue, Room 706A, University of Miami, Miami, FL,; E-mail: Sbhattacharya@med.miami.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 January 15. Published in final edited form as:Biochemistry. 2008 January 15; 47(2): 817-825. doi:10.1021/bi701517m. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript peroxidase that are elevated in the aqueous humour of...

Levuglandins (LGs) and isolevuglandins (isoLGs, also called "isoketals" or "isoKs") are extraordinarily reactive products of cyclooxygenase and free radical-induced oxidation of arachidonates. We now report the detection in vivo and quantitative analysis of LG/isoLG adducts that incorporate the amino group of phosphatidylethanolamines (PEs) into LG/isoLGhydroxylactams. Notably, LC-MS/MS detection of these hydroxylactams is achieved with samples that are an order of magnitude smaller and sample processing is much simpler and less timeconsuming than required for measuring protein-derived LG/isoLG-lysyl-lactams. A key feature of our protocol is treatment of biological phospholipid extracts with phospholipase A 2 to generate mainly 1-palmitoyl-2-lysoPE-hydroxylactams from heterogeneous mixtures of phospholipids with a variety of acyl groups on the 2-position. Over 160% higher mean levels of LG/isoLG-PEhydroxylactam (P < 0.001) were detected in liver from chronic ethanol-fed mice (32.4 ± 6.3 ng/g, n = 6) compared to controls (12.1 ± 1.5 ng/g, n = 4), and mean levels in plasma from patients with agerelated macular degeneration (5.2 ± 0.4 ng/ml, n = 15) were elevated ~53% (P < 0.0001) compared to healthy volunteers (3.4 ± 0.1 ng/ml, n = 15). Just as LG/isoLG-protein adducts provide a dosimeter of oxidative injury, this study suggests that LG/isoLG-PE-hydroxylactams are potential biomarkers for assessing risk for oxidative stress-stimulated diseases.

Protein modifications in which the ε-amino group of lysyl residues is incorporated into a 2-(ω-carboxyethyl)pyrrole (CEP) are mediators of age-related macular degeneration (AMD). They promote both angiogenesis into the retina (“wet AMD”) and geographic retinal atrophy (“dry AMD”). Blood levels of CEPs are biomarkers for clinical prognosis of the disease. To enable mechanistic studies of their role in promoting AMD, e.g., through the activation of B- and T-cells, interaction with receptors, or binding with complement proteins, we developed an efficient synthesis of CEP derivatives, that is especially effective for proteins. The structures of tryptic peptides derived from CEP-modified proteins were also determined. A key finding is that 4,7-dioxoheptanoic acid 9-fluorenylmethyl ester reacts with primary amines to provide 9-fluorenylmethyl esters of CEP-modified proteins that can be deprotected in situ with 1,8-diazabicyclo[5.4.0]undec-7-ene without causing protein denaturation. The introduction of multiple CEP-modifications with a wide variety of CEP:protein ratios is readily achieved using this strategy.

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.

Low density lipoprotein contains traces of biologically active platelet-activating factor (PAF)-like ether phosphatidylcholines (PCs). These oxidatively truncated alkylacylphosphatidylcholines (OxPAFs) are presumably formed through the oxidative truncation of 1-alkyl-2-polyunsaturated fatty acyl PCs. We now report that a diverse structural variety of OxPAFs are generated in small unilamellar vesicles (SUVs) upon myeloperoxidase (MPO)-promoted autoxidation of ether PCs that incorporate linoleoyl, arachidonyl, or docosahexaenoyl groups at the sn-2 position. Total syntheses are reported that confirm the identities of the new OxPAFs and will facilitate the evaluation of their biologically important chemistry and activities. Especially noteworthy is the formation of OxPAFs containing g-hydroxyalkenal functionality. Analogous oxidatively truncated diacylphosphatidylcholines are biologically important because they and their more oxidized derivatives are strong ligands for the scavenger receptor CD36. Furthermore, their covalent adduction with proteins can interfere with protein function or generate biologically active carboxyalkylpyrrole derivatives. We now find a profound influence of membrane composition on the stability of OxPAFs. In the presence of a polyunsaturated diacyl PC, the linoleic acid ester of 2-lysophosphatidylcholine, MPO induces the oxidation of aldehydes to carboxylic acids and the further oxidative truncation of g-hydroxyalkenals. Remarkably, these reactions do not occur readily with MPO in SUVs composed entirely of saturated diacyl-PCs. A mechanistic rationale is presented that can account for this dichotomy.-Chen, X., W. Zhang, J. Laird, S. L. Hazen, and R. G. Salomon. Polyunsaturated phospholipids promote the oxidation and fragmentation of g-hydroxyalkenals: formation and reactions of oxidatively truncated ether phospholipids. J. Lipid Res. 2008. 49: 832-846.