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: 7594059
• 负责人: Ignacio R Rodriguez
• 金额: $ 173.08万
• 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/7594059
• 关键词: 7-ketocholesterol; Age related macular degeneration; Aging; Agonist; Apoptosis; Area; Arterial Fatty Streak; Back; Basic Science; Biology; Carbon; Cell Nucleus; Cells; Cholesterol; Cholesterol Esters; Choroidal Neovascularization; Chronic; Class; Condition; Copper; Cytochrome P450; Cytosol; Deposition; Disease; Docosahexaenoic Acids; Drosophila genus; Drug Metabolic Detoxication; Enzymes; Extravasation; Fatty Acids; Fertility; Genes; Goals; HDL receptor; Hydroxylation; Inorganic Sulfates; Iron; Lipids; Lipoproteins; Liver; Location; Longevity; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Mediating; Membrane; Messenger RNA; Metabolic Pathway; Metabolism; Methionine; Mitochondria; Mixed Function Oxygenases; Mus; Object Attachment; Pathogenesis; Pathway interactions; Personal Satisfaction; Photoreceptors; Physiological; Play; Polyunsaturated Fatty Acids; Predisposition; Primates; Process; Protein Overexpression; Proteins; Rattus; Research; Research Project Grants; Retina; Retinal; Retinal Diseases; Role; Side; Sterols; Synapses; Toxic effect; Transgenic Organisms; Unspecified or Sulfate Ion Sulfates; age related; albino rat; cell type; cytochrome c; cytotoxic; cytotoxicity; feeding; ganglion cell; interest; lipid transport; macula; methionine sulfoxide reductase; mitochondrial membrane; oxidation; oxidized lipid; oxidized low density lipoprotein; particle; promoter; receptor; research study; restoration; retinal rods; reverse cholesterol transport; sulfation; sulfotransferase; transcription factor; uptake

Recently, research done at the MRDS has demonstrated that the retina uptakes much of its lipids from circulating low density lipoproteins (LDL) via LDL-receptors in the RPE and choriocapillaris (4). Inside the RPE the lipids are processed and delivered as HDL-like particles to HDL-receptors (SR-BI and SR-BII) in the photoreceptor cells (3) and other cells of the inner retina. We have demonstrated that the retina expresses all of the main genes involved in the well-known systemic reverse cholesterol transport pathway. The retina has adapted this pathway to sustain its own particular needs for lipid transport and turnover by controlling the expression and location of the different transporters and receptors in the pathway (3). 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. We are presently pursuing this project by generating transgenic rats with RPE and photoreceptor-specific targeted overexpression and knockdowns of selective genes. Another related area of interest is the formation of oxidized lipids and their cytotoxicity. One particular molecule, 7-ketocholesterol, 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-esters in LDL deposits but is also known to be readily formed by the photooxidation of cholesterol. The toxicity of 7-ketocholesterol is due to its ability to form destabilizing crystals in mitochondrial membranes leading to cytochrome-c leakage and apoptosis. Our studies have found small amounts of 7-ketocholesterol in normal primate retina mostly associated with oxidized LDL deposits in Bruchs membrane and choriocapillaris (unpublished results). However, in light-damaged albino rats 7-ketocholesterol levels greatly increase throughout the retina especially in the ganglion cells, RPE and photoreceptor inner segments (unpublished results). This suggests that photooxidation is a plausible mechanism for generating 7-ketocholesterol in the retina. Moreover, this also suggests that chronic mitochondrial damage due to 7-ketocholesterol formation could be a factor in aging diseases of the retina. We have also found that 7-ketocholesterol can induce VEFG release from cultured RPE cells and from the RPE and choriocapillaris of rats injected with oxidized LDL. This may be a potential mechanism for some forms of AMD that are characterized by choroidal neovascularization. Our experiments have also shown that lipoproteins like LDL can be readily photoxidized forming a mixture of oxidized lipids of similar composition to those found in atherosclerotic plaques. The polyunsaturated fatty acids are also of interest because they are components of numerous lipid classes and are highly oxidizable at their double bonds. Docosahexaenoic acid (DHA) is of particular interest since it comprises approximately 50% of the lipids in the outer segment membranes. This fatty acid is highly susceptible to photooxidation and its transport and metabolism in the retina is not fully understood. Enzymes that protect the mitochondria from oxidative damage or from 7-ketocholesterol toxicity are also a major area of interest. The cytochrome P450 CYP27A1 is a sterol hydroxylase capable of hydroxylating 7-ketocholesterol and is highly expressed in the mitochondria of rod photoreceptors and RPE (1). 7-Ketocholesterol when hydroxylated at the side chain carbons loses its toxicity (1). Sulfation of 7-ketocholesterol is another potential mechanism of detoxification and very small amounts of sulfated 7-ketocholesterol have been dettected in primate retina. Although hydroxylation seems to be the major detoxification pathway for 7-ketocholesterol in the retina, sterol sulfation may be an important regulatory feed-back mechanism to provide antagonists to the liver X-receptors (LXRs). LXRs are transcription factors that regulate many of the genes in the reverse cholesterol transport pathway and are highly expressed in the retina. It is well known that oxysterols are agonists of LXRs while some sulfated sterols are antagonists. We are presently searching for a retinal-expressed sulfotransferase capable of sulfating 7-ketocholesterol under physiological conditions. Another group of protective enzymes of interest to the MRDS are the methionine sulfoxide reductases (MSRs). These groups of enzymes (MSRAs and MSRBs) convert oxidized methionines in proteins back to methionine often resulting in the restoration of lost function. Overxpression of MSRA in the fruit fly increased their lifespan and fertility. Mice lacking MSRA have greatly reduced lifespans and are highly susceptible to oxidative damage. Previous studies on MSRA suggest that it play a key role in aging and age-related disease. We have found that MSRA is highly expressed in the macular RPE (2). The retina contains high levels of both MSRA and MSRB activities (2). We also discovered that MSRA is controlled by two distinct promoters. One promoter (P1) makes an mRNA whose product is targeted to the photoreceptor synaptic mitochondria and the other promoter (P2) makes two mRNAs whose products are targeted to the cytosol and nucleus. The P2 promoter is highly expressed in RPE cells. We are presently characterizing the P2 promoter and have identified several relevant transcription factors involved in regulating its expression. 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.

最近，在MRDS进行的研究表明，视网膜通过RPE和Choriocapillaris中的LDL受体摄取的低密度脂蛋白（LDL）吸收了许多脂质（4）。在RPE内部，将脂质用于光感受器细胞（3）和内部视网膜的其他细胞中的HDL样颗粒（SR-BI和SR-BII）作为HDL样颗粒（SR-BI和SR-BII）进行处理并传递。我们已经证明，视网膜表达了涉及众所周知的全身反向胆固醇转运途径的所有主要基因。视网膜通过控制不同转运蛋白和受体在途径中的表达和位置来适应这种途径，以维持其对脂质运输和离职的特殊需求（3）。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。目前，我们正在通过产生带有RPE的转基因大鼠和具有感光受体特异性的靶向过表达和敲低选择性基因的转基因大鼠。 感兴趣的另一个相关领域是形成氧化脂质及其细胞毒性。一种特定的分子，7-酮胆脂酯醇特别感兴趣，因为已知它对各种细胞类型具有高度的细胞毒性，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LDL沉积物中胆固醇溶解的氧化形成的，但也已知很容易通过胆固醇的光氧化而形成。 7-酮固醇​​的毒性是由于其在线粒体膜中形成不稳定晶体的能力，导致细胞色素-C泄漏和凋亡。我们的研究发现，在正常的灵长类动物视网膜中，少量的7-酮胆脂脂溶胶主要与Bruchs膜和绒毛膜乳房中的氧化LDL沉积有关（未发表的结果）。然而，在损坏的白化大鼠中，在整个视网膜中，7-酮胆脂酯水平大大增加，尤其是在神经节细胞中，RPE和感光器的内部段（未发表的结果）。这表明光氧化是在视网膜中产生7-酮固醇​​的合理机制。此外，这还表明，由于7-酮胆脂酯形成引起的慢性线粒体损伤可能是视网膜衰老疾病的一个因素。我们还发现，7-酮胆固醇可以诱导培养的RPE细胞以及注射氧化LDL的大鼠的RPE和绒毛膜中的VEFG释放。这可能是某些以脉络膜新生血管形成特征的AMD的潜在机制。 我们的实验还表明，像LDL这样的脂蛋白可以很容易地被光氧化，形成与动脉粥样硬化斑块中的氧化脂质的混合物。多不饱和脂肪酸也很感兴趣，因为它们是许多脂质类别的组成部分，并且在其双键时具有高度氧化。 Docosahexaenoic Acid（DHA）特别令人感兴趣，因为它包含外部段膜中约50％的脂质。这种脂肪酸高度容易受到光氧化的影响，其在视网膜中的运输和代谢尚不完全了解。 保护线粒体免受氧化损伤或从7-酮胆固醇毒性中的酶也是一个主要感兴趣的领域。细胞色素P450 CYP27A1是一种能够羟基化7-酮胆固醇的固醇羟化酶，在Rod感光体和RPE的线粒体中高度表达（1）。 7-酮胆固醇在侧链碳氧化液时失去其毒性（1）。 7-酮胆固醇的硫酸化是排毒的另一种潜在机制，而在灵长类动物视网膜中已将少量的硫化7-酮胆脂脂溶液介绍。尽管羟基化似乎是视网膜中7-酮胆脂脂酯的主要解毒途径，但固醇硫酸化可能是为肝X受体（LXR）提供拮抗剂的重要调节饲料机制。 LXR是调节反向胆固醇转运途径中许多基因的转录因子，并在视网膜中高度表达。众所周知，氧蛋白酶是LXR的激动剂，而某些硫酸化的固醇是拮抗剂。目前，我们正在寻找能够在生理条件下硫化7-酮胆脂酯硫醇的视网膜表达的磺胺转移酶。 MRDS感兴趣的另一组保护酶是甲硫氨酸还原酶（MSR）。这些酶（MSRAS和MSRB）将蛋白质中的氧化蛋氨酸转化为蛋氨酸，通常会导致恢复功能的恢复。 MSRA在果蝇中的压倒性提高了它们的寿命和生育能力。缺乏MSRA的小鼠的寿命大大降低，并且非常容易受到氧化损伤。先前对MSRA的研究表明，它在衰老和与年龄有关的疾病中起关键作用。我们发现MSRA在黄斑RPE（2）中高度表达。视网膜含有高水平的MSRA和MSRB活动（2）。我们还发现，MSRA由两个不同的启动子控制。一个启动子（P1）制作的mRNA，其产物针对感光体突触线粒体，另一个启动子（P2）产生了两个mRNA，其产物靶向细胞质和核。 P2启动子在RPE细胞中高度表达。我们目前正在表征P2启动子，并确定了调节其表达的几个相关转录因子。 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。