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 该研究项目将检验以下假设：Tortilin是在 钙化主动脉瓣疾病（CAVD）通过促进成肌纤维细胞样胶原蛋白产生瓣膜 间质细胞（VIC）表型和VIC衍生的细胞外囊泡（EV）钙化的诱导。这 从未研究过Tortilin在CAVD中的作用。我们公正的基于网络的系统生物学 方法发现，Tortilin网络高度显着接近p38 MAPK蛋白网络。在 另外，表达表达硅蛋白的VIC的单细胞RNA测序确定了主要生物学的富集 途径，包括细胞骨架组织，囊泡运输和钙化，因此表明 Tortilin通过诱导VICS中的肌纤维细胞样表型，纤维化和钙化，参与CAVD。 以前未知的Tortilin功能。本研究将探索托丁蛋白在主动脉瓣中的作用 在我们的机械研究中，钙化并关注这些关键途径。特定目标1将测试 在体内假设分类蛋白会加速主动脉瓣中纤维质的反应。这些实验会 在使用Tortilin缺陷型小鼠（复合突变体）的钙化主动脉狭窄的小鼠模型中进行 最近在PIS实验室建立的菌株和含有最小钙化的人主动脉瓣膜 从CAVD患者获得。在分子成像的控制下，这些传单的部分 将解剖并使用代表早期CAVD（例如，纤维化，微钙化）进行分析。在 此外，我们将采用创新方法来检测EV衍生的微钙化和VIC 表型，包括密度依赖性扫描电子显微镜（DD-SEM），高分辨率 显微镜，纳米颗粒跟踪分析，3D-Bioprinted水凝胶平台，蛋白质组学，单细胞 分析和复杂的网络分析。特定的目标2将在体外测试假说，即托利蛋白 通过促进VIC肌纤维细胞样表型，胶原蛋白的产生，介导VIC纤维钙化反应， 电动汽车的释放和矿化；新形成的胶原蛋白纤维内电动汽车的进一步聚集 导致形成微钙化。我们提出，托丁蛋白会诱导VIC纤维钙化反应 而遗传缺失的遗传缺失将减少胶原蛋白的产生并阻碍形成 人类维克斯和小鼠的微钙化。为了促进鼠标数据的临床翻译，我们将采用 来自CAVD患者的人类原发性vics和主动脉瓣标本。这些互补研究 将通过检查Tortilin在早期CAVD中的作用来推进该领域。从长远来看，从中的发现 项目将确定驱动CAVD的新型分子决定因素，并定义了急需的新目标 这种毁灭性疾病的患者的疗法。

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