Project 4 - Mechanisms of pyrophosphate dysregulation

项目4摘要 焦磷酸盐失调的机制。羟基磷灰石在骨骼和牙齿中的沉积是一种谨慎的 旨在平衡无机焦磷酸盐(PPI)浓度的协调生化过程 有效的钙化抑制剂和局部浓度的磷酸盐(PI)，钙化促进剂，以 建立适当的PPI/PI比率，以使受调控的组织矿化发生。当我们有一个很好的 了解控制生理性骨骼和牙齿矿化的分子，有限的数据存在于 这些机制的改变是否会导致眼睛、大脑和大脑的异位钙化 老年性黄斑变性(AMD)、阿尔茨海默病(AD)等老年性疾病的血管构筑 和动脉粥样硬化。AMD的临床特征是存在被称为玻璃体的细胞外沉积， 位于视网膜色素上皮(RPE)和脉络膜微血管之间的区域，称为 黄斑区的Bruch膜。在正常老化的眼睛中也可以观察到与 局部炎症和黄斑区大量大玻璃体的存在是一个重大风险。 发展AMD的因素。虽然还没有完全反映人类病理生理学的小鼠模型 AMD、Abcc6-/-和Enpp1-/-小鼠模型及推广 婴儿期动脉钙化(GACI)，不仅表现为脑血管钙化，还表现为脑血管钙化。 眼睛中的钙化，因此这些小鼠模型可以作为了解玻璃体的替代物 队形。在这个PO1组件中，我们将测试PPI/PI比率失调的总体假设 是老年病中异位钙化的病理生理学基础，我们的研究主要集中在 眼睛钙化。鉴于组织非特异性碱性磷酸酶(TNAP)是主要的焦磷酸酶 控制PPI/PI比值，TnAP可能成为预防/改善玻璃体疾病的有效药物靶点 队形。因此，本项目4的目标，旨在与其他PO1相互支持 成分，旨在探讨PPI失调在眼部钙化中的致病作用 在体内操纵PPI的产生和降解。我们将调查PPI/PI比率是否 通过产生和功能控制(在脉络膜内皮细胞、RPE细胞或两者中) 一种特殊类型的细胞外小泡，称为基质小泡(MVS)，在那里羟基磷灰石开始形成 在生理性骨骼/牙齿矿化过程中，或者如果这个比例是通过病理生理控制的 单独上调TNAP活性。我们还将进行药理学概念验证研究，旨在 通过调节全身和局部PPI的产生和降解来影响眼内钙化 浓度。完成这些研究，在这一多学科小组的努力范围内，将导致 对AMD、AD和血管钙化相互关联的病理生理学的新见解，并将验证 治疗/预防表现为异位钙化的临床需求未得到满足的可用药途径 这些高度流行的衰老疾病。

PROJECT 4 SUMMARY Mechanisms of pyrophosphate dysregulation. Hydroxyapatite deposition in bones and teeth is a carefully orchestrated biochemical process aimed at balancing the concentrations of inorganic pyrophosphate (PPi), a potent calcification inhibitor, and the local concentrations of phosphate (Pi), a calcification promoter, to establish a proper PPi/Pi ratio to enable regulated tissue mineralization to take place. While we have a good understanding of what molecules control physiological skeletal and dental mineralization, limited data exists as to whether alterations in those same mechanisms lead to ectopic calcification in the eyes, brain, and vasculature in diseases of aging such as age-related macular degeneration (AMD), Alzheimer's disease (AD) and atherosclerosis. AMD is clinically characterized by the presence of extracellular deposits known as drusen, located between the retinal pigment epithelium (RPE) and the choroidal microvasculature in the area known as the Bruch’s membrane in the macular region. Drusen is also observed in the normal aging eye associated with local inflammation, and the presence of a high number of large drusen in the macular region is a significant risk factor for developing AMD. While there are no murine models that fully reflect the pathophysiology of human AMD, the Abcc6-/- and the Enpp1-/- mouse models of pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) respectively, manifest not only vascular and brain calcification but also calcifications in the eyes and therefore these mouse models can be used as surrogates to understand drusen formation. In this PO1 component we will test the overarching hypothesis that dysregulation of the PPi/Pi ratio underlies the pathophysiology of ectopic calcification in diseases of aging, focusing our studies primarily on eye calcification. Given that tissue-nonspecific alkaline phosphatase (TNAP) is the major pyrophosphatase controlling the PPi/Pi ratio, TNAP may be a useful druggable target for the prevention/amelioration of drusen formation. Thus, the goals of this Project 4, designed to be mutually supportive with the other PO1 components, are to probe the causative role of PPi dysregulation in eye calcification by genetically manipulating the production and degradation of PPi in vivo. We will investigate if the PPi/Pi ratio is being controlled (either in the choroid endothelium, RPE cells or both compartments) via the production and function of a special type of extracellular vesicles known as matrix vesicles (MVs), where hydroxyapatite starts to form during physiological skeletal/dental mineralization, or if this ratio is controlled via the pathophysiological upregulation of TNAP activity alone. We will also conduct pharmacological proof-of-concept studies aimed at affecting calcification in the eye via modulating the production and degradation of systemic and local PPi concentrations. Completion of these studies, within the context of this multi-disciplinary team effort, will lead to novel insights into the interrelated pathophysiology of AMD, AD and vascular calcification and will validate a druggable pathway to address the unmet clinical need of treating/preventing ectopic calcification manifesting in these highly prevalent diseases of aging.