Quantitative Analysis of Aging Retina

描述(申请人提供)：老年性黄斑病变(ARM)是西方社会无法治愈的老年人视力丧失的主要原因。早期ARM的特点是视网膜下有鲜为人知的脂肪沉积。我们认为：与动脉粥样硬化性心血管疾病一样，ARM涉及局部细胞对血管内膜中含载脂蛋白B的脂蛋白的滞留的反应，但含有载脂蛋白B的脂蛋白来自视网膜色素上皮(RPE)。我们发现，在正常老化的Bruchs膜(BRM)中存在酯化的胆固醇和80-100 nm的固体颗粒，玻璃体和基底沉积物中含有胆固醇和apoB，RPE中含有apoB和微粒体甘油三酯转移蛋白(MTTP)的mRNA和蛋白，这是脂蛋白分泌细胞的标志。结构性RPE脂蛋白途径的扰动是形成高胆固醇病变的一种可能的机制。RPE脂蛋白的一个看似合理的功能是清除吞噬的光感受器外节中的脂肪酸。我们的首要任务是证明RPE脂蛋白途径的存在，并了解其正常功能。在人眼中，我们将使用电子显微镜、免疫组织化学、密度梯度超速离心法和酶类脂分析来描述分离的玻璃体和BRM中的脂蛋白颗粒。在培养的ARPE-19细胞中，我们将确定最佳的载脂方法，以便对分泌颗粒的成分进行化学和超微结构表征，并在体外确定调节MTTP活性对RPE脂蛋白组装和分泌的影响。在小鼠中，我们将研究在正常和明亮的光线下，系统地给予特定和有效的MTTP抑制剂对RPE中光感受器吞噬后细胞内油滴形成的影响。我们的结果将在评估ARM和动脉粥样硬化性心血管疾病在细胞外脂蛋白积累和脂蛋白分泌细胞生物学方面共享机制的程度上具有价值。这一信息是确定是否应该考虑将改变肝脂蛋白产生的治疗用于治疗早期ARM所必需的。

DESCRIPTION (provided by applicant): Age-related maculopathy (ARM) is the leading cause of untreatable vision loss among the elderly in Western society. Early ARM features poorly understood fatty deposits under the retina. We propose that: ARM, like atherosclerotic cardiovascular disease, involves local cellular response to the retention of an apolipoprotein (apo) B-containing lipoprotein in a vascular intima, with the twist that the apoB-containing lipoprotein derives from retinal pigment epithelium (RPE). We show that esterified cholesterol and 80-100 nm solid particles are present in normal aged Bruch's membrane (BrM), drusen and basal deposits contain cholesterol and apoB, and the RPE contains mRNA and protein for apoB and microsomal triglyceride transfer protein (MTTP), the hallmark of a lipoprotein secreting cell. Perturbation of a constitutive RPE lipoprotein pathway is a plausible mechanism for the formation of cholesterol-enriched lesions. A plausible function for an RPE lipoprotein is to clear fatty acids from phagocytosed photoreceptor outer segments. Our first priority is proving that an RPE lipoprotein pathway exists and learning its normal function. In human eyes, we will describe lipoprotein particles in isolated drusen and in BrM, using electron microscopy, immunohistochemistry, density gradient ultracentrifugation, and enzymatic lipid assays. In cultured ARPE-19 cells, we will determine the optimal method for lipid-loading so that the components of secreted particles can be characterized chemically and ultrastructurally, and we will determine the effect of modulating MTTP activity on RPE lipoprotein assembly and secretion in vitro. In mice, we will study the effect of systemic administration of specific and potent inhibitors of MTTP on the formation of intracellular oil droplets in RPE following photoreceptor phagocytosis in normal and bright light. Our results will be valuable in assessing the extent which ARM and atherosclerotic cardiovascular disease share mechanisms with regard to extracellular lipoprotein accumulation and the cell biology of lipoprotein secreting cells. This information is required to determine if treatments that modify hepatic lipoprotein production should be considered for treating early ARM.