Characterization of ENTPD1 as a drug target in PXE

这项研究将集中在可遗传异位矿化的原型--弹性假黄瘤(PXE)上 精神错乱。PXE中异常的磷酸钙沉积导致特征性的皮肤表现 眼部并发症可能导致视力减退和失明，如 以及心血管受累，包括肾源性高血压，间歇性跛行， 胃肠道动脉，早期心肌梗死和中风。目前还没有有效的治疗PXE和 因此，疾病在确诊后会继续发展。PXE由ABCC6基因突变引起，ABCC6基因编码 ABCC6，一种主要在肝脏表达的外排转运蛋白。我们小组阐明了病理生理学。 PXE的基础，通过显示肝细胞中的ABCC6介导ATP释放到血液循环中。 释放的ATP被胞外核苷酸酶ENPP1细胞外转化为1个AMP和1个分子 强大的矿化抑制剂无机焦磷酸(PPI)的分子。PXE患者存在缺陷 ABCC6具有较低的PPI血浆浓度，这解释了它们在软结缔组织中的进行性钙化。 血浆PPI的稳态是通过参与ATP释放的蛋白质的复杂网络来维持的，并且 代谢释放的核苷酸的胞外核苷酸酶。ENPP1在这方面至关重要，因为它的活动 是血浆中存在的所有PPI的基础。PXE患者血浆中~40%的残存PPI被排出 由独立于ABCC6的细胞释放的ATP。细胞外的ATP也受其他外源物质代谢的影响。 核苷酸酶和ENPP1在循环中最重要的竞争对手是ENTPD1，它转换ATP 1个AMP分子和2个无机磷酸盐分子。重要的是，ENPP1和ENTPD1具有 相反的影响细胞外PPI的动态平衡，因此，异位钙化。在这项研究中，我们 建议检验抑制ENTPD1促进细胞外PPI形成和 预防PXE患者的异位钙化我们制定了两个具体目标来验证我们的假设： (1)深入研究ENPP1和ENTPD1之间的竞争如何影响胞外PPI 体外动态平衡和(2)测定ENTPD1对血浆PPI浓度和异位的影响 Abcc6-/-小鼠体内的钙化。这些计划以坚实的初步数据为基础，它们利用了 我们系开发和鉴定了一种特性良好的小鼠模型--Abcc6-/-小鼠-- 概述了人类PXE的临床、组织病理学、超微结构和遗传学特征。 总的来说，我们利用体外和体内模型系统进行的最先进的研究有望提供 抑制ENTPD1恢复血浆PPI水平的关键临床前信息 PXE，随后抑制异位钙化。我们的发现预计不仅将为发展提供信息 对PXE的药物治疗，也对其他异位钙化疾病，没有有效或 目前已经有了特定的治疗方法。

This research will focus on pseudoxanthoma elasticum (PXE), the prototype of heritable ectopic mineralization disorders. Aberrant calcium phosphate deposition in PXE results in characteristic cutaneous findings which signify the potential for development of ocular complications leading to loss of visual acuity and blindness, as well as cardiovascular involvement, including nephrogenic hypertension, intermittent claudication, bleeding from gastrointestinal arteries, early myocardial infarct and stroke. There is no effective treatment for PXE and the disease therefore progresses after diagnosis. PXE is caused by mutations in the ABCC6 gene which encodes ABCC6, an efflux transporter expressed primarily in the liver. Our group elucidated the pathophysiology underlying PXE, by showing that ABCC6 in hepatocytes mediates release of ATP into the blood circulation. Released ATP is extracellularly converted by the ecto-nucleotidase ENPP1 into 1 molecule of AMP and 1 molecule of the potent mineralization inhibitor inorganic pyrophosphate (PPi). PXE patients bearing defective ABCC6 have low PPi plasma concentrations, explaining their progressive calcification of soft connective tissues. Plasma PPi homeostasis is maintained through a complex network of proteins involved in ATP release, and ecto-nucleotidases metabolizing the released nucleotides. ENPP1 is crucial in this respect, as its activity underlies all PPi present in plasma. The ~40% of residual PPi present in plasma of PXE patients is formed out of ATP released by cells independent of ABCC6. Extracellular ATP is also subject to metabolism by other ecto- nucleotidases and the most important competitor of ENPP1 in the circulation is ENTPD1, which converts ATP into 1 molecule of AMP and 2 molecules of inorganic phosphate. Importantly, ENPP1 and ENTPD1 have opposing effects on extracellular PPi homeostasis and, consequently, ectopic calcification. In this study we propose to test the hypothesis that inhibition of ENTPD1 enhances extracellular PPi formation and prevents ectopic calcification in PXE patients We have developed two Specific Aims to test our hypothesis: (1) Thoroughly characterize how competition between ENPP1 and ENTPD1 affects extracellular PPi homeostasis in vitro and (2) Determine the effect of ENTPD1 on plasma PPi concentrations and ectopic calcification in Abcc6-/- mice in vivo. These plans are based on solid preliminary data, and they take advantage of a well-characterized mouse model developed and characterized in our department – the Abcc6-/- mouse –, which recapitulates the clinical, histopathologic, ultrastructural and genetic features of PXE in humans. Collectively, our state-of-the-art studies utilizing both in vitro and in vivo model systems are expected to provide critical preclinical information of the therapeutic potential of ENTPD1 inhibition to restore plasma PPi levels in PXE, with subsequent inhibition of ectopic calcification. Our findings are expected to not only inform development of pharmacologic treatments for PXE, but also for other ectopic calcification disorders, for which no effective or specific therapy is currently available.