The vessel wall as a paracrine engine in TGF beta-induced aortic aneurysm

血管壁作为 TGFβ 诱导的主动脉瘤的旁分泌引擎

• 批准号: 8752550
• 负责人: Elena Gallo MacFarlane
• 金额: $ 11.4万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8752550
• 关键词: Ablation; Address; Affect; Aneurysm; Angiotensin II Type 1 Receptor Blockers; Aortic Aneurysm; Arteries; Award; Binding; Biochemical; Bioinformatics; Blood; Blood Vessels; Cardiac; Cells; Clinical; Complement; Confusion; Development; Development Plans; Disease; Disease Progression; Dissection; Embryo; Equilibrium; Event; Fibrosis; Flow Cytometry; Fostering; Genes; Genetic; Goals; Heart; Immune; Immunofluorescence Immunologic; In Situ; In Vitro; Inherited; Knowledge; Lead; Left; Ligands; Loeys-Dietz Syndrome; Losartan; Malignant Neoplasms; Mediator of activation protein; Mentors; Methods; Modeling; Molecular; Mus; Mutation; Neural Crest; PTPRC gene; Paracrine Communication; Pathogenesis; Pathology; Pathway interactions; Patients; Phase; Phenotype; Plant Roots; Population; Post-Translational Protein Processing; Process; Proteomics; RNA analysis; Research Proposals; Role; Rupture; Signal Transduction; Signaling Molecule; Site; Smooth Muscle Myocytes; Techniques; Testing; Therapeutic; Tissues; Training; Transforming Growth Factor beta; Transforming Growth Factors; Uncertainty; Up-Regulation; Work; base; career development; cell type; genetic manipulation; in vivo; induced pluripotent stem cell; kinase inhibitor; loss of function mutation; mouse model; paracrine; prevent; progenitor; receptor; research study; response; skills

DESCRIPTION(provided by applicant): An aneurysm is an abnormal dilation of an artery that, if left untreated, can lead to fatal tear or rupture. TGFβ signaling upregulation is observed in aortic tissue from patients affected by hereditary aneurysm disorders, such as Loeys-Dietz syndrome (LDS), that are caused by heterozygous loss-of-function mutations in genes encoding positive regulators of the TGFβ pathway. These paradoxical findings have caused considerable controversy and have called into question the use of TGFβ antagonists as therapy for these disorders. The goal of this proposal is to identify the cellular and biochemical mechanisms that cause TGFβ signaling overdrive in LDS. We propose to test a non-cell autonomous mechanism based on the observation that LDS mutations in either of the two genes that encode TGFβ receptor subunits have discordant effects on the signaling capacity of aortic cell types that are adjacent in the vessel wall but derived from different embryonic progenitors. Specifically, these mutations leave "high responder" cells such as cardiac neural crest-derived vascular smooth muscle cells (CNC-VSMCs) relatively unaffected, while they impair the TGFβ response of more vulnerable "low responder" cells, such as second heart field-derived VSMCs (SHF-VSMCs). The overall hypothesis of this proposal is that compensatory events aimed at restoring normal signaling in "low responder" cells, such as upregulation of TGFβ ligand expression, will induce TGFβ signaling overdrive in "high responder" cells, ultimately culminating in the development of aneurysm. In order to test this hypothesis, I will work with my primary mentor, Dr. Dietz, an expert on aneurysm pathogenesis, and with my co-mentor, Dr. Van Eyk, an expert in clinical proteomics applications, to address the first two aims. In Aim1, I will define the cellular mediators of TGFβ paracrine overdrive by analyzing the TGFβ response of control and LDS SHF-VSMCs and CNC-VSMCs, and the repertoire of paracrine factors secreted by these cells both in vivo and in vitro. In Aim 2, I will functionally test the TGFβ paracrine overdrive model by pharmacological and genetic manipulations. Specifically, I will assess whether global TGFβ antagonism or cellspecific reduction of TGFβ signaling in "high responder" cells, but not "low responder" cells, ameliorates aneurysm progression in LDS mice. During this phase of the award I will become proficient in the field of proteomics and bioinformatics by working with Dr. Van Eyk's lab and by participating in formal coursework. The new skills, techniques, and knowledge that I will gain will foster my transition to the R00 phase, in which I will independentl address Aim 3 and Aim 4. In Aim 3 I propose to identify the molecular determinants of lineagespecific TGFβ responses by using proteomics-based techniques and conventional biochemical methods to analyze, both in vitro and in vivo, the differential expression and/or post-translational modifications of modulators of TGFβ signaling in "high responder" and "low responder" cells. In Aim 4, I intend to elucidate the contribution of CD45+ immune cells to TGFβ signaling paracrine overdrive. I will characterize the phenotype of the CD45+ immune cells infiltrate that is present at the site of aneurysm in LDS mouse models and assess whether TGFβ ligand secreted by these cells contributes to exacerbation of pathology. I am confident that the training and career development plan described in this proposal will enable me to elucidate the mechanisms of excessive TGFβ signaling in LDS and will provide critical information for the understanding of cell-type specific TGFβ-responses. The studies described in this proposal have the potential to increase our understanding of other TGFβ-related disorders, such as fibrosis and cancer, in which signaling imbalances between interacting cell-types may drive pathology.

描述（由申请人提供）：动脉瘤是动脉的异常扩张，如果不及时治疗，可能会导致致命的撕裂或破裂。在患有遗传性动脉瘤疾病（例如 Loeys-Dietz 综合征 (LDS)）的患者的主动脉组织中观察到 TGFβ 信号传导上调，这种疾病是由编码 TGFβ 途径正调节因子的基因杂合性功能丧失突变引起的。这些矛盾的发现引起了相当大的争议，并对使用 TGFβ 拮抗剂治疗这些疾病提出了质疑。该提案的目标是确定导致 LDS 中 TGFβ 信号过度驱动的细胞和生化机制。我们建议测试一种非细胞自主机制，该机制基于以下观察结果：编码 TGFβ 受体亚基的两个基因中的任何一个的 LDS 突变对血管壁相邻但源自不同胚胎祖细胞的主动脉细胞类型的信号传导能力具有不一致的影响。具体来说，这些突变使“高反应”细胞，如心脏神经嵴来源的血管平滑肌细胞（CNC-VSMC）相对不受影响，而它们损害了更脆弱的“低反应”细胞，如第二心野来源的VSMC（SHF-VSMC）的TGFβ反应。该提议的总体假设是，旨在恢复“低反应”细胞中正常信号传导的补偿事件，例如 TGFβ 配体表达的上调，将诱导“高反应”细胞中 TGFβ 信号过度驱动，最终导致动脉瘤的发展。为了检验这一假设，我将与我的主要导师、动脉瘤发病机制专家 Dietz 博士以及临床蛋白质组学应用专家 Van Eyk 博士合作，解决前两个目标。在 Aim1 中，我将通过分析对照和 LDS SHF-VSMC 和 CNC-VSMC 的 TGFβ 反应，以及这些细胞在体内和体外分泌的旁分泌因子库来定义 TGFβ 旁分泌过度驱动的细胞介质。在目标 2 中，我将通过以下方式对 TGFβ 旁分泌过度驱动模型进行功能测试 药理学和基因操作。具体来说，我将评估“高反应”细胞（而不是“低反应”细胞）中的整体 TGFβ 拮抗作用或 TGFβ 信号传导的细胞特异性减少是否可以改善 LDS 小鼠的动脉瘤进展。在该奖项的这一阶段，我将通过与 Van Eyk 博士的实验室合作并参加正式课程，精通蛋白质组学和生物信息学领域。我将获得的新技能、技术和知识将促进我向 R00 阶段的过渡，在此阶段我将独立解决目标 3 和目标 4。在目标 3 中，我建议通过使用基于蛋白质组学的技术和常规生化方法来分析体外和体内的差异表达和/或翻译后修饰，从而确定谱系特异性 TGFβ 反应的分子决定因素。 “高反应”和“低反应”细胞中 TGFβ 信号传导的调节剂。在目标 4 中，我打算阐明 CD45+ 免疫细胞对 TGFβ 信号旁分泌过度驱动的贡献。我将描述 LDS 小鼠模型中动脉瘤部位存在的 CD45+ 免疫细胞浸润的表型，并评估这些细胞分泌的 TGFβ 配体是否会导致病理恶化。我相信，本提案中描述的培训和职业发展计划将使我能够阐明 LDS 中过度 TGFβ 信号传导的机制，并为理解细胞类型特异性 TGFβ 反应提供关键信息。该提案中描述的研究有可能增加我们对其他 TGFβ 相关疾病的理解，例如纤维化和癌症，其中相互作用的细胞类型之间的信号失衡可能会驱动病理学。