REACTIVE INTERMEDIATES OF OXIDATIVE LIPID FRAGMENTATION

氧化脂质断裂的反应中间体

• 批准号: 9114118
• 负责人: Robert Gerd Salomon
• 金额: $ 38.36万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9114118
• 关键词: 4 hydroxynonenal; Accounting; Acids; Age related macular degeneration; Alcohols; Aldehydes; Amines; Apoptosis; Appearance; Atrophic; Basic Science; Binding; Biochemistry; Biological; Blood; Blood Platelets; Bone Marrow Cells; Brain Injuries; Catalysis; Cell Culture Techniques; Cell membrane; Cells; Cellular Membrane; Chemistry; Choroidal Neovascularization; Culture Media; Cytosol; Diffuse; Diffusion; Docosahexaenoic Acids; Endothelial Cells; Equus caballus; Ethanol; Ethanolamines; Extracellular Matrix; Generations; Glean; Glutathione; Health; Human; In Vitro; Individual; Inflammatory; Injury; Interferons; Interleukin-17; Investigation; Lactones; Leukotrienes; Light; Lipid Bilayers; Lipids; Location; Measures; Membrane; Membrane Proteins; Modeling; Modification; Neurons; Pathogenesis; Pathway interactions; Patients; Phospholipids; Photoreceptors; Physiological; Pilot Projects; Post-Translational Protein Processing; Proteins; Rattus; Reaction; Research; Retina; Retinal; Scheme; Signal Pathway; Signal Transduction; Site; T-Lymphocyte; TLR1 gene; TLR2 gene; TP53 gene; Testing; Therapeutic immunosuppression; Tissues; Tube; Vascular Endothelial Growth Factors; adduct; angiogenesis; base; clinically significant; deacylation; design; extracellular; geographic atrophy; in vivo; insight; macrophage; monolayer; novel therapeutics; oxidation; oxidized lipid; prevent; receptor; retinal damage; retinal rods

 DESCRIPTION (provided by applicant): Docosahexaenoate phospholipids (DHA-PLs) are uniquely abundant in retinal and neuronal cells. Our basic research led to the discoveries that oxidation of DHA-PLs results in the generation of carboxyethylpyrrole (CEP) modifications of proteins and ethanolamine phospholipids whose levels are elevated in retinas and blood from individuals with age-related macular degeneration (AMD). Subsequent research revealed that CEPs stimulate angiogenesis found in the choroidal neovascularization of "wet" AMD through a vascular endothelial growth factor-independent mechanism involving Toll-like receptor (TLR)2 signaling. CEPs also contribute to the global retinal atrophy of "dry" AMD by IFNᵞ and IL-17-producing CEP-specific T cells that promote M1 polarization of macrophages in the retina. Our most recent studies revealed that oxidized DHA-PLs release HOHA-lactone that can dissociate from cell membranes and react with proteins to generate CEPs, previously only known to be produced by direct reaction of an oxidized DHA-PLs with proteins. We now propose studies of HOHA-lactone chemistry and transport through cell membranes and monolayers to evaluate the likelihood that its escape from DHA-rich membranes of photoreceptor rod cell disks can produce CEPs in locations remote from the site of membrane oxidation. This may contribute to the clinically significant elevated levels of CEPs we discovered in the blood of individuals with AMD and it may account for CEP generation in the blood of rats upon light-induced oxidative injury of their retinas. We will examine the possibility that HOHA-lactone can enter cells and generate CEP modifications of intracellular proteins that can bind with and activate intracellular receptors such as platelet TLR9. Studies of HOHA-lactone glutathione (GSH) Michael adduct biochemistry will test the hypotheses that this adduct can serve as a Trojan horse that transports a CEP precursor out of cells, and that in conjunction with ALD-catalyzed reduction, can prevent CEP formation. Inspired by the biological activities found previously for adducts of other oxidized lipids with GSH, e.g. leukotrienes, pilot studies were conducted with GSH adducts of HOHA-lactone that revealed that submicromolar concentrations of GSH-HOHA-lactone and the alcohol produced by reduction of this aldehyde stimulate proliferation and tube formation by HUVEC cells. New cell biological studies are proposed to investigate the effects of HOHA-lactone, its GSH adducts and the CEP modifications of proteins and ethanol-amine phospholipids derived from the HOHA-lactone on primary human RPE cells, bone marrow-derived macrophages, and primary choroidal endothelial cells, including studies on the signaling pathways leading to the biological effects. The potential utility of the mechanistic information to be gleaned from the in vitro and in vivo studies proposed is exemplified by new insights recently developed suggesting that immunosuppressive therapy might be effective for ameliorating the retinal damage of "dry" AMD caused by a CEP-induced T-cell promoted invasion of the retina by inflammatory macrophages and their CEP-potentiated activation.

 描述（由申请人提供）：二十二碳六烯酸磷脂（DHA-PL）在视网膜和神经元细胞中非常丰富。我们的基础研究发现，DHA-PL的氧化导致蛋白质和乙醇胺磷脂的羧乙基吡咯（CEP）修饰的产生，其水平在患有年龄相关性黄斑变性（AMD）的个体的视网膜和血液中升高。随后的研究表明，CEP通过涉及Toll样受体（TLR）2信号传导的血管内皮生长因子非依赖性机制刺激在"湿性" AMD的脉络膜新血管形成中发现的血管生成。CEP还通过产生IFN β和IL-17的CEP特异性T细胞促进视网膜中巨噬细胞的M1极化而导致"干性" AMD的整体视网膜萎缩。我们最近的研究表明，氧化的DHA-PL释放HOHA-内酯，其可以从细胞膜上解离并与蛋白质反应以产生CEP，以前只知道通过氧化的DHA-PL与蛋白质的直接反应产生CEP。我们现在提出的HOHA-内酯的化学和运输通过细胞膜和单层的研究，以评估其从DHA丰富的感光杆细胞盘膜逃逸的可能性，可以产生CEP的位置远离膜氧化的网站。这可能有助于我们在AMD个体血液中发现的临床显著升高的CEP水平，并且这可能解释了在光诱导的视网膜氧化损伤后大鼠血液中CEP的产生。我们将研究HOHA-内酯进入细胞并对细胞内蛋白产生CEP修饰的可能性，这些蛋白可以与细胞内受体结合并激活细胞内受体 如血小板TLR9。HOHA-内酯谷胱甘肽（GSH）迈克尔加合物生物化学的研究将测试假设，这种加合物可以作为一个特洛伊木马，运输CEP前体的细胞，并与ALD催化还原，可以防止CEP的形成。受先前发现的其他氧化脂质与GSH的加合物（例如白三烯）的生物活性的启发，用HOHA-内酯的GSH加合物进行了初步研究，结果表明亚微摩尔浓度的GSH-HOHA-内酯和通过还原该醛产生的醇刺激HUVEC细胞的增殖和管形成。提出了新的细胞生物学研究来研究HOHA-内酯、其GSH加合物以及HOHA-内酯衍生的蛋白质和乙醇胺磷脂的CEP修饰对原代人RPE细胞、骨髓源性巨噬细胞和原代人视网膜色素上皮细胞的影响。脉络膜内皮细胞，包括导致生物学效应的信号通路的研究。机械信息的潜在效用， 从所提出的体外和体内研究中收集到的这些信息通过最近开发的新见解来举例说明，该新见解表明免疫抑制疗法可能有效地改善由CEP诱导的T细胞促进的炎性巨噬细胞对视网膜的侵袭及其CEP增强的活化引起的"干性" AMD的视网膜损伤。