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: 7734604
• 负责人: Ignacio R Rodriguez
• 金额: $ 109.82万
• 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/7734604
• 关键词: 7-ketocholesterol; Age; Age related macular degeneration; Aging; Area; Arterial Fatty Streak; Back; Basic Science; Biology; CYP46A1 gene; Carbon; Cell Nucleus; Cells; Cholesterol; Cholesterol Esters; Choroid Hemorrhage; Choroidal Neovascularization; Chronic; Class; Complex; Copper; Cytochromes; Cytosol; Deposition; Disease; Docosahexaenoic Acids; Drug Metabolic Detoxication; Enzymes; Extravasation; Fatty Acids; Genes; Goals; HDL receptor; Hydroxylation; IL8 gene; In Vitro; Inorganic Sulfates; Interleukin-6; Iron; Lipids; Lipoproteins; Liver; Location; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Mediating; Membrane; Messenger RNA; Metabolic Pathway; Metabolism; Methionine; Mitochondria; Object Attachment; Pathogenesis; Pathway interactions; Photoreceptors; Photosensitizing Agents; Play; Polyunsaturated Fatty Acids; Positioning Attribute; Predisposition; Primates; Process; Property; Proteins; Research; Research Project Grants; Retina; Retinal; Retinal Diseases; Role; SULT2B1; Sp1 Transcription Factor; Synapses; Time; Toxic effect; Unspecified or Sulfate Ion Sulfates; Vascular Endothelial Growth Factors; age related; albino rat; cell type; cytotoxic; cytotoxicity; ganglion cell; in vivo; interest; lipid transport; macula; methionine sulfoxide reductase; oxidation; oxidized lipid; oxidized low density lipoprotein; particle; promoter; receptor; research study; restoration; reverse cholesterol transport; sulfation; sulfotransferase; transcription factor; uptake

Research performed 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. 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 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 its own particular needs by controlling the expression and location of the different lipoproteins, transporters and receptors. 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. 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 lipoprotein deposits. It can also be made by photooxidation of cholesterol in the presence of a suitable photosensitizing agent. The mechanism(s) of toxicity of 7-ketocholesterol is complex. 7-ketocholesterol is known to form micro-crystals in membranes causing destabilization and possible leakage. However, 7-ketocholesterol also has potent pharmacological properties causing the induction of VEGF, IL-6 and IL-8. This seems to occur via the liver X receptors (LXRs) and the transcription factor Sp1. Our studies have found small amounts of 7-ketocholesterol in normal primate retina mostly associated with oxidized LDL deposits in Bruchs membrane and choriocapillaris. In light-damaged albino rats 7-ketocholesterol levels greatly increase throughout the retina especially in the ganglion cells, RPE and photoreceptor inner segments. 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. The chronic deposition of oxidized LDL in the choriocapillaris and Bruchs membrane may be a potential mechanism for choroidal neovascularization and the forms of AMD associated with choroidal hemorrhage. 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. We have found that either sulfation or hydroxylation can neutralize the toxicity of 7-ketocholesterol in vitro. The MRDS has focused particularly on the cytochrome P450s, CYP27A1 and CYP46A1. CYP27A1 is abundant in the photoreceptor and RPE mitochondria and hydroxylates 7-ketocholesterol mostly at the 26-27 carbon positions. CYP46A1 is present only in trace amounts in the retina and is unlikely to play any significant role in 7-ketocholesterol hydroxylation. Moreover, after extensively searching for hydroxylated forms of 7-ketocholesterol by LCMS, we have only found traces in both in vivo and in vitro experiments. This suggests that hydroxylation is an unlikely form of 7-ketocholesterol detoxification. Sulfation by the sulfotransferase enzyme SULT2B1 was also investigated but this enzyme is only present in trace amounts in the retina and 7-ketocholesterol sulfate is not detectable in retinal extracts. There are several other sulfotransferases in the retina but the MRDS is not actively investigating their function at this time. 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. Previous studies on MSRA suggest that it plays a key role in aging and age-related disease. We have found that MSRA is highly expressed in the macular RPE. The retina contains high levels of both MSRA and MSRB activities. 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 have partially characterized 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.

在MRDS进行的研究表明，视网膜通过RPE和绒毛膜中的LDL受体吸收了循环低密度脂蛋白（LDL）的大部分脂质。在RPE内部，将脂质作为HDL样颗粒进行处理，并将其输送到光感受器细胞和内部视网膜的其他细胞中的HDL受体（SR-BI和SR-BII）。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。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。 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。该氧化酚是由铜蛋白沉积中胆固醇溶解剂的铜和/或铁介导的氧化形成的。在存在合适的光敏剂的情况下，胆固醇的光氧化也可以通过。 7-酮胆脂质的毒性机制很复杂。已知7-酮胆固醇会在膜上形成微晶，导致稳定和可能的泄漏。然而，7-酮胆脂质也具有有效的药理特性，导致VEGF，IL-6和IL-8的诱导。这似乎是通过肝X受体（LXR）和转录因子SP1发生的。我们的研究发现，在正常的灵长类动物视网膜中，少量的7-酮胆脂脂醇与Bruchs膜和绒毛膜的氧化LDL沉积物有关。在损坏的白化病大鼠中，在整个视网膜中，7-酮胆脂脂酯水平大大增加，尤其是在神经节细胞，RPE和感光细胞内部段。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。氧化的LDL在脉络膜毛细血管和Bruchs膜中的长期沉积可能是脉络膜新生血管形成的潜在机制以及与脉络膜出血相关的AMD形式。 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。我们发现，硫酸化或羟基化可以中和7-酮胆脂脂的体外毒性。 MRD特别关注细胞色素P450S，CYP27A1和CYP46A1。 CYP27A1在光感受器和RPE线粒体中很丰富，羟基盐酸盐7-酮胆固醇主要在26-27碳位置。 CYP46A1仅存在于视网膜中的痕量中，并且不太可能在7-酮胆固醇羟基化中起任何重要作用。此外，在广泛搜索LCM的7-酮胆脂酯醇的羟基化形式之后，我们仅在体内和体外实验中都发现了痕迹。这表明羟基化是7-酮胆固醇解毒的一种不太可能的形式。 还研究了用硫代转移酶Sult2B1硫化的硫酸盐，但该酶仅在视网膜中以痕量存在于痕量中，而在视网膜提取物中无法检测到7-酮胆固醇硫酸盐。 视网膜中还有其他几个磺胺转移酶，但MRDS目前尚未积极研究其功能。 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。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中高度表达。视网膜含有高水平的MSRA和MSRB活动。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。我们已经部分表征了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。