Mechanism of human valve interstitial cell calcification

人体瓣膜间质细胞钙化机制

• 批准号: RGPIN-2017-05328
• 负责人: Schwertani, Adel
• 金额: $ 1.82万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711615
• 关键词: Mechanism; human; valve; interstitial; cell

Overall aims: The general aim of the current proposal is to determine mechanism of the human aortic valve interstitial cell (HAVIC) mineralization in relation to increases in lipoprotein(a)[Lp(a)] and oxidized phospholipids (OxPLs). Background and Rationale: Although historically HAVIC mineralization (calcification) thought to be a degenerative process, recent studies have pointed to other possible causes such as shear-stress, angiotensin system and hyperlipidemia. Lp(a) is composed of the glycoprotein apolipoprotein (a) [apo(a)] attached to apolipoprotein B-100, found in a particle highly similar to low density lipoprotein (LDL). Lp(a) also binds to OxPLs in plasma and up taken in tissue by scavenger receptors. Studies utilizing transgenic mice have supported a relationship between Lp(a) serum levels and mineralization of blood vessels. Although these studies point to a possible relationship between Lp(a) and vascular stiffness, only recent genetic population studies have pointed to an a potentially important role for Lp(a) in aortic valve calcification. How Lp(a) induces HAVICs mineralization, and the mechanisms involved remains to be determined. General Hypothesis: Lp(a) is uptaken by HAVICs by endocytosis or receptor binding, and subsequently decreasing cholesterol efflux and inducing mineralization and apoptosis through increased matrix vesicles (MVs) formation and reactive oxygen species (ROS). Interfering with Lp(a) and/or downstream signalling pathway would ameliorate HAVICs mineralization. Experimental design: We plan to determine the mechanism of: 1) Lp(a) uptake and/or binding in HAVICs? HAVICs express ABCA1, LDL-R, SRB-1 and LRP5/6. Here, we shall assess Lp(a) catabolism and cholesterol efflux in HAVICs. 2) Lp(a) induced HAVICs mineralization? When HAVICs undergo mineralization they produce a large number of calcium-phosphate containing matrix vesicles (MVs). Our preliminary data showed that treatment of HAVICs with Lp(a) significantly increased the number of MVs and expression of proteins and genes involved in the biogenesis of MVs accompanied by a significant reduction in Fetuin-A. Here, we plan to isolate and characterize these MVs using ultracentrifugation, NanoSight, flow cytometry and electron microscopy. We also plan to identify the functional profiles of these MVs using quantitative proteomics in order to interfere with the pathways of downstream mediators of Lp(a)-induced effects in HAVICs. 3) Lp(a)-induced HAVICs apoptosis. Here, we plan to determine the levels of ROS and monitor mitochondrial transmembrane potential in response to Lp(a). 4) In vivo effects of Lp(a) overexpression/inhibition and the mechanism involved. In the short term, the proposed work will determine the mechanism of HAVICs mineralization, and in the long term serves as an excellent platform for the training of graduate students and post-doctoral fellows.

总体目标：当前提案的总体目的是确定人主动脉瓣间质细胞（HAVIC）矿化与脂蛋白（a）[Lp（a）]和氧化磷脂（OxPL）增加相关的机制。 背景和依据：虽然历史上HAVIC矿化（钙化）被认为是一个退行性过程，但最近的研究指出了其他可能的原因，如剪切应力，血管紧张素系统和高脂血症。脂蛋白（a）是由糖蛋白载脂蛋白（a）[apo（a）]连接到载脂蛋白B-100，发现在一个颗粒高度相似的低密度脂蛋白（LDL）。Lp（a）也与血浆中的OxPL结合，并通过清道夫受体在组织中摄取。利用转基因小鼠的研究支持Lp（a）血清水平与血管矿化之间的关系。虽然这些研究指出Lp（a）和血管僵硬之间可能存在关系，但只有最近的遗传群体研究指出Lp（a）在主动脉瓣钙化中可能起重要作用。Lp（a）是如何诱导HAVIC矿化的，其机制尚不清楚。 一般假设：Lp（a）通过内吞作用或受体结合被HAVIC摄取，随后通过增加基质囊泡（MV）形成和活性氧（ROS）减少胆固醇流出并诱导矿化和凋亡。干扰Lp（a）和/或下游信号通路可改善HAVIC的矿化。 实验设计：我们计划确定以下机制： 1)HAVIC中Lp（a）的摄取和/或结合？HAVIC表达ABCA 1、LDL-R、SRB-1和LRP 5/6。在此，我们将评估HAVIC中的Lp（a）催化剂和胆固醇流出。 2)Lp（a）诱导HAVICs矿化？当HAVIC经历矿化时，它们产生大量含有磷酸钙的基质囊泡（MV）。我们的初步数据显示，用Lp（a）处理HAVIC显著增加了MV的数量以及参与MV生物发生的蛋白质和基因的表达，伴随着胎球蛋白-A的显著减少。在这里，我们计划使用超离心，NanoSight，流式细胞术和电子显微镜分离和表征这些MV。我们还计划使用定量蛋白质组学来鉴定这些MV的功能谱，以干扰HAVIC中Lp（a）诱导效应的下游介质的途径。 3)Lp（a）诱导HAVIC凋亡。在这里，我们计划确定ROS水平和监测线粒体跨膜电位响应Lp（a）。 4)Lp（a）过表达/抑制的体内效应及其机制 从短期来看，拟议的工作将确定HAVIC矿化的机制，从长远来看，将成为研究生和博士后研究员培训的良好平台。