REACTIVE INTERMEDIATES OF OXIDATIVE LIPID FRAGMENTATION

 描述(申请人提供)：二十二碳六烯酸磷脂(DHA-PLs)在视网膜和神经细胞中独一无二地丰富。我们的基础研究发现，DHA-pls的氧化导致羧乙基吡咯(CEP)修饰的蛋白质和乙醇胺磷脂的产生，这些修饰蛋白和乙醇胺磷脂在年龄相关性黄斑变性(AMD)患者的视网膜和血液中水平升高。随后的研究发现，CEPS通过Toll样受体(TLR)2信号转导的血管内皮生长因子非依赖性机制，刺激“湿性”AMD脉络膜新生血管的形成。CEP还通过干扰素ᵞ和产生IL-17的CEP特异性T细胞促进视网膜巨噬细胞M1极化，促进干性AMD的全球视网膜萎缩。我们最新的研究表明，氧化的DHA-pls释放HOHA-内酯，HOHA-内酯可以从细胞膜上解离并与蛋白质反应生成CEPs，以前已知的只有氧化的DHA-pls与蛋白质直接反应才能产生CEPs。我们现在建议研究HOHA-内酯的化学和通过细胞膜和单层的运输，以评估其从光感受器杆状细胞盘富含DHA的膜中逃逸的可能性，从而在远离膜氧化部位的位置产生CEP。这可能是我们在AMD患者血液中发现的CEP水平在临床上显著升高的原因之一，也可能是光诱导视网膜氧化损伤时大鼠血液中CEP产生的原因。我们将研究HOHA-内酯进入细胞并产生细胞内蛋白的CEP修饰的可能性，该修饰可以与细胞内受体结合并激活细胞内受体 如血小板TLR9。对HHA-内酯谷胱甘肽(GSH)Michael加合物生物化学的研究将检验这样的假设，即该加合物可以作为特洛伊木马将CEP前体输送到细胞外，并且与ALD催化的还原一起可以防止CEP的形成。受以前发现的其他氧化脂质与GSH加合物的生物活性的启发，例如白三烯，对HOHA-内酯的GSH加合物进行了初步研究，发现亚微摩尔浓度的GSH-HOHA-内酯以及该醛还原产生的酒精可刺激HUVEC细胞的增殖和管状形成。新的细胞生物学研究是为了研究HOHA-内酯及其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脉络膜内皮细胞的影响，包括导致生物效应的信号通路的研究。机械性信息的潜在用途 从体外和体内研究中收集到的是最近发展的新见解的例证，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细胞促进炎症巨噬细胞入侵视网膜及其CEP增强的激活而引起的干性AMD的视网膜损伤。

 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.