Lipid transport, oxidation and toxicity in the retina

视网膜中的脂质运输、氧化和毒性

• 批准号: 7968306
• 负责人: IGNACIO R RODRIGUEZ
• 金额: $ 130.15万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968306
• 关键词: 7-ketocholesterol; Age related macular degeneration; Aging; Aging-Related Process; Apolipoprotein E; Apolipoproteins B; Apoptosis; Area; Arterial Fatty Streak; Basic Science; Biology; Blood Circulation; Bruch' s basal membrane structure; CD36 gene; CETP gene; CYP46A1 gene; Carbon; Cell Death; Cells; Cessation of life; Cholesterol; Cholesterol Esters; Complex; Copper; Cultured Cells; Cytochrome P450; Deposition; Disease; Dose; Enzymes; Extravasation; Ferritin; Free Radicals; Goals; Health; Human; IL8 gene; Inflammation; Inflammatory; Interleukin-6; Iron; Lasers; Light; Lipids; Lipoproteins; Liver; Location; Low-Density Lipoproteins; Mediating; Membrane; Metabolism; Methionine; Mitochondria; Modeling; Monkeys; Necrosis; Neural Retina; PRDX3 peroxidase; Pathogenesis; Pathway interactions; Phosphatidylcholine-Sterol O-Acyltransferase; Photoreceptors; Predisposition; Process; Property; Proteins; Rattus; Reactive Oxygen Species; Research Project Grants; Retina; Retinal; Retinal Diseases; Role; SULT2B1; Side; Source; Stereoisomer; Testing; Toxic effect; Transition Elements; Vascular Endothelial Cell; Vascular Endothelial Growth Factors; albino rat; base; catalyst; cell type; cholesterol 24-hydroxylase; cytochrome c; cytokine; cytotoxic; cytotoxicity; ganglion cell; in vivo; interest; lipid transport; macula; meetings; methionine sulfoxide; methionine sulfoxide reductase; oxidation; oxidative damage; oxidized lipid; particle; prevent; receptor; sulfotransferase; uptake

The MRDS has shown that the retina uptakes circulating LDL and distributes the lipids to the different cellular layers. To perform this lipid uptake and transport, the retina expresses the same proteins used by the systemic "reverse cholesterol" pathway. The retina has adapted this pathway to its own particular needs by controlling the expression and location of the different lipoproteins, transporters and receptors. Monkey retina has been found to express apoA1, apoE, apoB, ABCA1, LDLR, SR-BI and II, CD36, CETP, and LCAT. The particular locations where these molecules are expressed within the retina suggest an internal lipid transport based on HDL-like lipid particles. We hypothesize that the retina requires a high turnover of lipids because of the high susceptibility of this class of molecules to oxidation and particularly to photooxidation. One of the main priorities of the MRDS is to identify the mechanism by which oxidized lipids, which may be highly toxic, are metabolized and excreted from the retina. Recently the MRDS has found that the retina contains significant levels of the highly toxic oxysterol, 7-ketocholesterol. This molecule is of particular interest because it is known to be highly cytotoxic to various cell types and is the major toxic component in atherosclerotic plaques. This oxysterol is formed by copper and/or iron mediated oxidation of cholesterol and cholesterol-esters in lipoprotein deposits. In the monkey retina 7-ketocholesterol is found in association with oxidized lipoprotein deposits in the choriocapillaris and Bruchs membrane. In photodamaged albino rats 7-ketocholesterol is found in areas of high mitochondrial content especially the RPE, photoreceptor inner segments and ganglion cells. Intermediates identified in these rats by LCMS indicate that the 7-ketocholesterol was formed via a free radical mediated mechanism. This mechanism requires a transition metal catalyst which is likely Fe+2. The source of the iron was not conclusively identified but light is known to cause the release of iron from ferritin and possibly cytochrome c. The source of the iron and mechanisms of its release are being actively investigated. In cultured human RPE and vascular endothelial cells the MRDS has found that 7-ketocholesterol is a potent inducer of VEGF and other cytokines including IL-6 and IL-8. The pharmacological properties of 7-ketocholesterol are complex and seem to be dose dependent. At low doses 7-ketocholesterol is pro-inflammatory while at higher doses it can induce cell death by necrosis or apoptosis depending on the cell type. In most cell types 7-ketocholesterol-induced inflammation is dependent on reactive oxygen species (ROS) formation. However, in cultured RPE-derived cells the pro-inflammatory pathway seems to be independent of ROS. 7-ketocholesterol is also known to form micro-crystals in membranes causing destabilization and possible leakage and/or breakdown. The MRDS is actively investigating the 7-ketocholesterol-mediated inflammatory and death pathways in the retina in vivo. In addition, the MRDS is testing the anti-angiogenic properties of certain antagonist to 7-ketocholesterol-mediated VEGF induction using a CNV rat model (laser treated). The metabolism of 7-ketocholesterol by the neural retina and RPE are another topic of interest to the MRDS. The retina expresses significant levels of the mitochondrial CYP27A1, a cytochrome P450 enzyme capable of hydrolylating 7-ketocholesterol at the side chain 27-carbon. This hydroxylated form of 7-ketocholesterol is non-toxic and lacks the pro-inflammatory properties of 7-ketocholesterol. However, repeated attempts at detecting hydroxylated metabolites of 7-ketocholesterol in retinal extracts have been unsuccessful. Two other enzymes capable of metabolizing 7-ketocholesterol have been investigated, the 24-cholesterol hydroxylases (CYP46A1) and the sulfotransferases (SULT2B1). Both of these enzymes are present in the retina but only in trace amounts and their potential metabolites are also undetectable. The retina seems to eliminate 7-ketocholesterol without metabolizing it. Preliminary evidence suggests transporters such as ABCG1 which is present in the basal aspect of the RPE may be involved in the elimination of 7-ketocholesterol. Once in circulation 7-ketocholesterol is known to be quickly metabolized by the liver. Another area of interest to the MRDS are anti-oxidative enzymes specially those that protect the mitochondria. In this regard we have investigated the retinal expression of peroxiredoxin 3 and the methionine sulfoxide reductases (MSRs). These are enzymes capable of reversing the oxidation of methionine in proteins and thus preserving function. There are two major forms of MSRs categorized by their stereospecificity on methionine sulfoxide (MetO). The MSRAs recognize the Met(S)O and the MSRBs recognize the Met(R)O stereoisomers. These enzymes have been shown to be important in preserving the health of cells and in the aging process. The MSRs are highly expressed in the retina especially in the macular region. These enzymes protect cultured cells from multiple forms of oxidative damage including damage caused by 7-ketocholesterol. The MRDS is actively investigating the role of these enzymes in preventing the light-mediated iron release from protein like ferritin. In summary the MRDS is pursuing several basic research projects investigating lipid transport, oxidation and protective mechanisms with the goal of obtaining a better understanding of the processes involved in aging and the pathogenesis mechanisms of diseases like the age-related macular degenerations.

MRD已表明，视网膜吸收循环LDL并将脂质分配到不同的细胞层。 为了执行这种脂质摄取和运输，视网膜表达了与系统性“反向胆固醇”途径使用的相同蛋白质。视网膜通过控制不同脂蛋白，转运蛋白和受体的表达和位置，使这一途径适应其自身的特殊需求。猴子视网膜已被发现表达apoa1，apoe，apob，abca1，ldlr，sr-bi和ii，cd36，cetp和lcat。这些分子在视网膜内表达的特定位置表明基于HDL样脂质颗粒的内部脂质转运。我们假设视网膜需要较高的脂质周转，因为这类分子对氧化，尤其是对光氧化的敏感性很高。 MRDS的主要优先事项之一是确定氧化脂质的机制（可能是剧毒的氧化脂质）是从视网膜中代谢和排泄的。 最近，MRDS发现视网膜中含有显着水平的剧毒氧蛋白酶7-酮胆脂酯。该分子特别令人感兴趣，因为已知它对各种细胞类型具有高度细胞毒性，并且是动脉粥样硬化斑块中的主要有毒成分。该氧化酚是由铜和/或铁介导的脂蛋白沉积中胆固醇和胆固醇 - 溶解剂的氧化形成的。在猴子视网膜中，发现7-酮降脂脂酯与脉络膜毛细血管和Bruchs膜中的氧化脂蛋白沉积物相关。在受损伤的白化病大鼠中，7-酮胆脂脂溶解酚在高线粒体含量的区域，尤其是RPE，感光细胞内部片段和神经节细胞。 LCMS在这些大鼠中鉴定出的中间体表明，7-酮胆脂酯是通过自由基介导的机制形成的。该机制需要Fe+2的过渡金属催化剂。铁的来源未得到最终识别，但已知光引起铁蛋白和可能的细胞色素c释放。正在积极研究铁和释放机制的来源。 在培养的人RPE和血管内皮细胞中，MRDS发现7-酮胆脂脂酯是VEGF和其他细胞因子的有效诱导剂，包括IL-6和IL-8。 7-酮固醇​​的药理特性很复杂，似乎依赖于剂量。在低剂量下，7-酮胆脂脂是促炎性的，而在较高剂量的情况下，它可以根据细胞类型诱导坏死或凋亡诱导细胞死亡。在大多数细胞类型中，7-酮胆脂酯诱导的炎症取决于活性氧（ROS）形成。但是，在培养的RPE衍生细胞中，促炎性途径似乎与ROS无关。还已知7-酮胆固醇在膜上形成微晶，导致不稳定以及可能的泄漏和/或分解。 MRD正在积极研究体内视网膜中7-酮胆脂酯介导的炎症和死亡途径。此外，MRDS使用CNV大鼠模型（激光处理）测试某些拮抗剂对7-酮胆脂酯介导的VEGF诱导的抗血管生成特性。 神经视网膜和RPE对7-酮胆脂酯的代谢是MRDS感兴趣的另一个话题。视网膜表达了明显水平的线粒体CYP27A1，这是一种细胞色素P450酶，能够在27-碳的侧链在侧链的7-酮胆脂脂酯溶液。这种7-酮固醇​​的这种羟基化形式无毒，缺乏7-酮胆固醇的促炎特性。然而，重复尝试检测视网膜提取物中7-酮胆固醇的羟化代谢产物的尝试尚未成功。已经研究了另外两种能够代谢7-酮胆酯醇的酶，分别是24-胆固醇羟化酶（CYP46A1）和磺胺转移酶（SULT2B1）。这两种酶都存在于视网膜中，但仅在痕量中，其潜在代谢产物也无法检测到。视网膜似乎可以消除7-酮胆脂酯，而不会代谢。初步证据表明，RPE基础方面存在的ABCG1等转运蛋白可能与消除7-酮胆固醇有关。循环后，已知7-酮胆脂酯会被肝脏迅速代谢。 MRD的另一个感兴趣的区域是抗氧化酶，特别是保护线粒体的酶。在这方面，我们研究了过氧蛋白3的视网膜表达和甲硫氨酸还原酶（MSR）。这些是能够逆转蛋白质中蛋氨酸并因此保留功能的酶。 MSR有两种主要形式，由它们在蛋氨酸亚氧化物（Meto）上的立体特异性分类。 MSRA识别MET（S）O，MSRB识别Met（R）O立体异构体。 这些酶已被证明在保留细胞健康和衰老过程中很重要。 MSR在视网膜中高度表达，尤其是在黄斑区域。这些酶可以保护培养的细胞免受多种形式的氧化损伤，包括由7-酮胆脂脂造成的损伤。 MRD正在积极研究这些酶在防止蛋白质像铁蛋白等蛋白质释放的铁中的作用。 总之，MRD正在追求几个基础研究项目，研究脂质运输，氧化和保护机制，目的是更好地了解衰老涉及的过程以及与年龄相关的黄斑变性等疾病的发病机制。