Mechanism of human valve interstitial cell calcification

总体目标：当前提案的总体目标是确定与脂蛋白(A)[Lp(A)]和氧化磷脂(Oxpls)增加有关的人主动脉瓣间质细胞(HAVIC)矿化的机制。背景和原理：虽然历史上认为HAVIC矿化(钙化)是一个退行性过程，但最近的研究指出了其他可能的原因，如切应力、血管紧张素系统和高脂血症。Lp(A)是由结合在载脂蛋白B-100上的糖蛋白apo(A)[apo(A)]组成的，在一种与低密度脂蛋白(LDL)高度相似的颗粒中发现。Lp(A)还与血浆中的Oxpls结合，并通过清道夫受体在组织中摄取。利用转基因小鼠进行的研究支持了血清Lp(A)水平与血管矿化之间的关系。尽管这些研究指出了Lp(A)与血管僵硬之间可能的关系，但只有最近的遗传群体研究才指出Lp(A)在主动脉瓣钙化中可能起着重要作用。Lp(A)是如何诱导HAVICs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HAVICs摄取，随后通过增加基质小泡(MVS)的形成和活性氧(ROS)的形成，减少胆固醇外流，诱导矿化和细胞凋亡。干扰Lp(A)和/或下游信号通路将改善HAVICs的矿化。*实验设计：我们计划确定：*1)HAVIC摄取和/或结合Lp(A)的机制？HAVICs表达ABCA1、LDL-R、SRB-1和LRP5/6。在这里，我们将评估HAVICs的Lp(A)分解代谢和胆固醇外流。*2)Lp(A)诱导HAVICs矿化？HAVICs在矿化过程中会产生大量含钙磷酸盐的基质小泡(MVS)。我们的初步数据显示，用LP(A)治疗HAVICs后，MVS的数量和参与MVS生物发生的蛋白质和基因的表达显著增加，同时胎球蛋白-A显著减少。在这里，我们计划使用超速离心法、纳米显微镜、流式细胞术和电子显微镜来分离和鉴定这些微血管。我们还计划利用定量蛋白质组学来鉴定这些MVS的功能，以便干扰Lp(A)诱导HAVICs中作用的下游介质的途径。在这里，我们计划测定ROS水平并监测线粒体跨膜电位对Lp(A)的响应。*4)Lp(A)过表达/抑制的体内效应及其涉及的机制。*短期内，拟议的工作将确定HAVICs的矿化机制，长期而言，将成为培养研究生和博士后研究员的良好平台。

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.