New driver of fibrosis and calcification in CAVD

项目摘要 这项研究项目将检验山梨素是纤维钙化反应的关键调节因子的假设。 肌成纤维细胞样胶原生成瓣膜促进钙化性主动脉瓣病 间质细胞(VIC)表型和VIC来源的细胞外小泡(EVS)钙化的诱导。这个 山梨素在CAVD中的作用从未被研究过。我们不偏不倚的网络系统生物学 方法发现，Sortilin网络与p38 MAPK蛋白网络高度显著接近。在……里面 此外，表达山梨素的VICs的单细胞RNA测序证实了主要的生物富集物 途径，包括细胞骨架组织，囊泡运输和钙化，因此表明 山梨素通过诱导VICs肌成纤维细胞样表型、纤维化和钙化参与CAVD。 以前未知的山梨素的功能。本研究将探讨山梨素在主动脉瓣中的作用。 并在我们的机制研究中重点关注这些关键途径。《特定目标1》将测试 体内假说：山梨素加速了主动脉瓣的纤维钙化反应。这些实验将 在钙化性主动脉狭窄小鼠模型中使用复合突变体sortilin缺乏的小鼠进行实验 最近在PIS实验室中发现的菌株和含有最低钙化的人主动脉瓣叶 取自CAVD患者。在分子成像的控制下，这些小叶的部分 代表早期CAVD的(如纤维化、微钙化)将被解剖并用于我们的分析。在……里面 此外，我们将采用创新的方法检测EV来源的微钙化和VIC 表型，包括密度依赖扫描电子显微镜(DD-SEM)，高分辨率 显微镜、纳米颗粒跟踪分析、3D生物打印水凝胶平台、蛋白质组学、单细胞 分析和复杂网络分析。《特定目标2》将在体外验证这一假说 通过促进VIC肌成纤维细胞样表型，胶原蛋白的产生， EVS的释放和矿化；EVS在新形成的胶原纤维中的进一步聚集 导致微钙化的形成。我们认为山梨素可诱导VIC纤维钙化反应 而山梨素的基因缺失会减少胶原的产生，延缓糖尿病的形成 人类受害者和小鼠体内的微钙化。为了促进老鼠数据的临床翻译，我们将使用 人的原发VICS和CAVD患者的主动脉瓣标本。这些互补性研究 将通过研究山梨素在早期CAVD中的作用来推动这一领域的发展。从长远来看，这一发现 该项目将确定驱动CAVD的新分子决定因素，并为急需的 治疗这种毁灭性疾病的方法。

Project Summary This research project will test the hypothesis that sortilin is a key regulator of fibrocalcific responses in calcific aortic valve disease (CAVD) through promotion of myofibroblast-like collagen producing valvular interstitial cells (VIC) phenotype and induction of VIC-derived extracellular vesicle (EVs) calcification. The role of sortilin in CAVD has never been investigated. Our unbiased network-based systems biology approach found that sortilin network is highly significantly close to the p38 MAPK protein network. In addition, single cell RNA sequencing of sortilin-expressing VICs identified enrichment of major biological pathways, including cytoskeletal organization, vesicle transport and calcification, thus suggesting that sortilin participates in CAVD by inducing myofibroblast-like phenotype, fibrosis and calcification in VICs, previously unknown functions of sortilin. The present study will explore the role of sortilin in aortic valve calcification and focus on these key pathways in our mechanistic studies. Specific Aim 1 will test the hypothesis in vivo that sortilin accelerates fibrocalcific responses in the aortic valve. These experiments will be performed in a mouse model of calcific aortic stenosis using sortilin-deficient mice, a compound mutant strain recently established in PIs laboratory, and human aortic valve leaflets containing minimal calcification obtained from patients with CAVD. Under control of molecular imaging, the portions of these leaflets representing early CAVD (e.g., fibrosis, microcalcification) will be dissected and used for our analyses. In addition, we will employ innovative methods for detection of EV-derived microcalcifications and VIC phenotypes, including density dependent scanning electron microscopy (DD-SEM), high-resolution microscopy, nanoparticle tracking analysis, 3D-bioprinted hydrogel platform, proteomics, single cell analyses, and complex network analyses. Specific Aim 2 will test the hypothesis in vitro that sortilin mediates VIC fibrocalcific response by promoting VIC myofibroblast-like phenotype, collagen production, and the release and mineralization of EVs; further aggregation of EVs within newly formed collagen fibers results in the formation of microcalcifications. We propose that sortilin induces VIC fibrocalcific responses and that genetic deletion of sortilin will decrease collagen production and retard the formation of microcalcifications in human VICs and mice. To facilitate clinical translation of mouse data, we will employ human primary VICs and aortic valve specimens from patients with CAVD. These complementary studies will advance the field by examining the role of sortilin in early CAVD. In the long-term, the findings from this project will identify novel molecular determinants that drive CAVD, and define new targets for much-needed therapies for patients with this devastating disorder.