New driver of fibrosis and calcification in CAVD

CAVD 纤维化和钙化的新驱动因素

• 批准号: 10374849
• 负责人: Elena Aikawa
• 金额: $ 58.72万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10374849
• 关键词: 3-Dimensional; Alkaline Phosphatase; Aortic Valve Stenosis; Arterial Fatty Streak; Arteries; Biological; Biological Markers; Blood Vessels; Breast Microcalcification; Cell Culture Techniques; Cells; Cessation of life; Clinical; Collagen; Collagen Fiber; Complex; Cytoskeletal Modeling; Cytoskeletal Proteins; Data; Development; Disease; Disease model; Early treatment; Economic Burden; Fibrosis; Genetic; Human; Hydrogels; Image; Immunohistochemistry; In Vitro; Injury; Laboratories; Left; Link; MAP Kinase Gene; Measures; Mediating; Medical; Messenger RNA; Microscopy; Modeling; Molecular; Molecular Biology; Monitor; Mus; Myofibroblast; National Heart, Lung, and Blood Institute; Network-based; Pathology; Pathway Analysis; Pathway interactions; Patients; Phenotype; Phosphorylation; Process; Production; Proteins; Proteome; Proteomics; Research Project Grants; Research Project Summaries; Resolution; Role; Scanning Electron Microscopy; Serum; Small Interfering RNA; Smooth Muscle Actin Staining Method; Sorting - Cell Movement; Specimen; Surgical Valves; System; Systems Biology; Testing; Therapeutic; Therapeutic Intervention; Time; Tissue Banks; Tissues; Ultrasonography; Vascular calcification; Work; aortic valve; aortic valve disorder; bioprinting; calcification; cell type; clinical imaging; clinical translation; cost; density; detection method; experimental study; extracellular vesicles; human tissue; imaging modality; in vivo; innovation; interstitial; interstitial cell; intervention cost; mineralization; molecular imaging; mouse model; mutant; nanoparticle; new therapeutic target; novel; osteogenic; osteogenic protein; p38 Mitogen Activated Protein Kinase; protein transport; receptor; repaired; response; single cell analysis; single-cell RNA sequencing; sortilin; targeted treatment; tool; trafficking; two-photon; valve replacement; vesicle transport; vesicular release

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.

项目摘要 这项研究项目将测试这一假设，分拣蛋白是一个关键的调节纤维钙化反应， 钙化性主动脉瓣疾病（CAVD）通过促进肌纤维母细胞样胶原蛋白产生瓣膜 间质细胞（维克）表型和诱导维克衍生的细胞外囊泡（EV）钙化。的 分拣蛋白在CAVD中的作用从未被研究过。我们公正的基于网络的系统生物学 方法发现分拣蛋白网络高度显著地接近p38 MAPK蛋白网络。在 此外，表达分拣蛋白的VIC的单细胞RNA测序鉴定了主要生物活性的富集。 途径，包括细胞骨架组织，囊泡运输和钙化，从而表明， 分拣蛋白通过诱导VIC中的成肌纤维细胞样表型、纤维化和钙化参与CAVD， 以前未知的功能。本研究将探讨分拣蛋白在主动脉瓣中的作用 钙化，并在我们的机制研究中关注这些关键途径。具体目标1将测试 假设分拣蛋白在体内加速主动脉瓣中的纤维钙化反应。这些实验将 在钙化性主动脉瓣狭窄的小鼠模型中使用分拣蛋白缺陷小鼠（复合突变体）进行 最近在PI实验室中建立的菌株，以及含有极轻微钙化的人主动脉瓣叶 从CAVD患者中获得。在分子成像的控制下，这些小叶的部分 代表早期CAVD（例如，纤维化、微钙化）将被解剖并用于我们的分析。在 此外，我们将采用创新的方法检测EV衍生的微钙化和维克 表型，包括密度依赖扫描电子显微镜（DD-SEM），高分辨率 显微镜，纳米粒子跟踪分析，3D生物打印水凝胶平台，蛋白质组学，单细胞 复杂网络分析。具体目标2将在体外检验分拣蛋白 通过促进维克成肌纤维细胞样表型，胶原蛋白产生， 以及EV的释放和矿化; EV在新形成的胶原纤维内的进一步聚集 导致微钙化的形成。我们认为分拣蛋白诱导维克血管内凝血纤维钙化反应 并且分拣蛋白的基因缺失将减少胶原蛋白的产生并延缓 在人类VIC和小鼠中的微钙化。为了促进小鼠数据的临床翻译，我们将采用 人原发性VIC和来自CAVD患者的主动脉瓣标本。这些补充研究 将通过研究sortilin在早期CAVD中的作用来推进该领域。从长远来看，这项研究的结果 该项目将确定驱动CAVD的新分子决定因素，并为急需的药物确定新的靶点。 治疗患有这种毁灭性疾病的患者。

项目成果

The Transcriptional Signature of Growth in Human Fetal Aortic Valve Development.

人类胎儿主动脉瓣发育生长的转录特征。

• DOI: 10.1016/j.athoracsur.2018.06.034
• 发表时间: 2018
• 期刊: The Annals of thoracic surgery
• 作者: GottliebSen,Danielle;Halu,Arda;Razzaque,Abdur;Gorham,JoshuaM;Hartnett,Jessica;Seidman,JonathanG;Aikawa,Elena;Seidman,ChristineE
• 通讯作者: Seidman,ChristineE