| We and others have postulated that oxidative protein modifications, including covalent crosslinks, may accumulate in trabecular meshwork (TM) and contribute to impaired aqueous outflow and primary open angle glaucoma (POAG). To test this hypothesis, human TM from normal and POAG donors was probed with antibodies for oxidative protein modifications. Studies using SDS PAGE and Western blotting showed elevated levels of covalent protein modifications derived from lipid oxidation products (iso[4]Levuglandin E2 (isoLGE 2) and 4-hydroxy-nonenal (HNE)), an advanced glycation end product (methylglyoxal), and a tryptophan oxidation product (3-hydroxykynurenine), in POAG compared to age-and-gender matched controls. Immunoprecipitation (IP) using iso[4]LGE 2 and HNE antibodies showed the presence of several apparently crosslinked proteins in POAG donor TM. 2D PAGE Western analyses, also showed proteins with altered molecular weights and pIs, implying modified and/or crosslinked proteins. Immunohistochemistry showed that iso[4]LGE2 and HNE modifications were localized to the TM. Thus, the study provides direct evidence for lipid-based oxidative modification of TM proteins in POAG. Additionally, increased levels of iso[4]LGE2 in the TM of the DBA/2J mouse model of glaucoma was also established and several putative crosslinked proteins were identified using IP and mass spectrometry. These results support a role for levuglandins (LGs) in POAG pathology. LGs are known to avidly bind with and crosslink proteins.; The inability of macrophages to processes modified proteins contributes to "foam cell" formation and atherosclerosis (AS). To identify the modified proteins in inflamed macrophages, lipopolysaccharide (LPS) stimulated macrophages were studied. LPS stimulation increased the levels of LGE2 modified proteins in macrophages. The modified proteins were identified by 2D PAGE and mass spectrometry. Many of the modified proteins are involved in cholesterol trafficking, gene expression, and/or lipid efflux. This suggests a role for LGE2 in AS. These observations inspired a pilot study to explore the possible role of LGs in LPS-induced inflammation of cornea in vivo. We found that LPS promotes the generation of LGs in cornea.; An efficient synthesis of the gamma-keto-alpha,beta-unsaturated alkenoic acid functional array present in some of the oxidatively truncated phospholipids, was achieved by oxidation of furyl precursors using NaClO 2. Additionally, detection and structural characterization of multiple HNE adducts onto lysine and histidine side-chain residues were accomplished using deuterated HNE and mass spectrometry. |
