The Role of THSD1 in Endothelial Cell Adhesion and Survival

THSD1 在内皮细胞粘附和存活中的作用

• 批准号: 8824389
• 负责人: Dong H Kim
• 金额: $ 7.7万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8824389
• 关键词: 13q14; Adherence; Adhesions; Aneurysmal Subarachnoid Hemorrhages; Animals; Apoptosis; Apoptotic; Basement membrane; Binding; Biological Assay; Biological Models; Blood; Blood Vessels; Brain hemorrhage; Cell Adhesion; Cell Death; Cell Survival; Cell-Cell Adhesion; Cell-Matrix Junction; Cells; Cellular Morphology; Cessation of life; Chromosomes; Clinical; Clinical Management; DNA Fragmentation; Disease; Drug Targeting; Early Diagnosis; Endothelial Cells; Endothelium; Environmental Risk Factor; Extracellular Matrix; Family; Family member; First Degree Relative; Focal Adhesion Kinase 1; Focal Adhesions; General Population; Genes; Genetic; Goals; Growth; Hemorrhage; Human Genome; Immunoblotting; Immunofluorescence Immunologic; Immunohistochemistry; In Vitro; Individual; Intracranial Aneurysm; Knock-out; Knockout Mice; Lead; Link; Manuscripts; Maps; Measures; Mediating; Molecular; Molecular Target; Monitor; Mus; Mutate; Mutation; Nonsense Mutation; Pathology; Pathway interactions; Patients; Phenotype; Phosphorylation; Play; Predisposition; Preparation; Proteins; Recording of previous events; Risk; Role; Rupture; Smooth Muscle Myocytes; Subarachnoid Hemorrhage; Survival Rate; Susceptibility Gene; Talin; Targeted Resequencing; TdT-Mediated dUTP Nick End Labeling Assay; Testing; Variant; Zebrafish; base; cerebral artery; cerebrovascular; clinical care; endothelial dysfunction; exome; exome sequencing; genetic linkage analysis; genetic variant; genome-wide linkage; improved; in vitro Model; induced pluripotent stem cell; insight; knock-down; mutant; novel; premature; prevent; protein function; public health relevance; treatment strategy

DESCRIPTION (provided by applicant): Rupture of an intracranial aneurysm (IA) is a common cause of subarachnoid hemorrhage (SAH), a leading cause of premature death. While environmental factors are believed to play a role in the formation and rupture of IAs, familial aggregation strongly suggests a genetic component. Ten to twenty percent of patients have a positive family history for IAs and aneurysmal SAH, and first-degree relatives have up to five-fold increased risk of developing disease compared to the general population. Many regions in the human genome have been linked to familial IA risk. However, disease-causing genetic variants have not been identified. Our long-term goals are to identify genes that when mutated predispose individuals to IAs, identify molecular pathways that lead to IA disease and develop treatment strategies to block IA growth. Using genomewide linkage and exome analyses on a large family, we identified the nonsense mutation p.R450* in THSD1 encoding thrombospondin-type 1 domain-containing protein 1 that is present in all 9 family members with IA. In addition, we identified 7 potential mutations in the same gene in other IA families. We observed that knocking down Thsd1 in zebrafish or mice causes spontaneous brain hemorrhage and premature death, suggesting a role for THSD1 in maintaining cerebrovascular integrity. THSD1 encodes a protein with unknown molecular function. Notably, THSD1 is expressed in endothelial cells, which form an important cell layer in cerebral arteries. Interestingly, a hallmark in IA pathology is the loss of the intact endothelium, suggesting a role for endothelial dysfunction in IA formation. THSD1 has been shown to bind talin, a focal adhesion protein that assists in the attachment of cells to the extracellular matrix. It has also been shown that when endothelial cells detach from underlying matrix, cells undergo programmed cell death. Since Thsd1 deficiency leads to hemorrhage, we hypothesize that THSD1 is involved in maintaining the integrity of the endothelium by promoting adhesion of endothelial cells to the underlying basement membrane and, consequently, mediating cell survival. In this application, we propose to use two in vitro model systems: 1) vascular endothelial and smooth muscle cells isolated from wild type and Thsd1-null mice; and 2) induced pluripotent stem cell (iPSC)-derived vascular endothelial and smooth muscle cells from wild type subjects and actual patients with the THSD1 p.R450* mutation. Using immunohistochemistry, immunoblotting, and functional assays, we aim to determine whether THSD1 localizes in focal adhesions, and if disruption of THSD1 would result in altered focal adhesion assembly, adherence to the extracellular matrix and cell survival. Successful demonstration of an abnormal phenotype will provide insight into the mechanism of THSD1-mediated IA formation and potentially identify molecular targets for preventative therapy.

描述(申请人提供)：颅内动脉瘤(IA)破裂是蛛网膜下腔出血(SAH)的常见原因，SAH是导致过早死亡的主要原因。虽然环境因素被认为在IAS的形成和破裂中发挥了作用，但家族聚集性强烈表明有遗传因素。10%到20%的患者有IAS和动脉瘤性SAH的阳性家族史，与普通人群相比，一级亲属发生疾病的风险增加了五倍。人类基因组中的许多区域都与家族性IA风险有关。然而，致病的基因变异还没有被确定。我们的长期目标是识别当突变时易患IAS的基因，识别导致IA疾病的分子途径，并开发阻止IA生长的治疗策略。通过对一个大家族的全基因组连锁和外显子组分析，我们在编码血栓反应蛋白1型结构域蛋白1的THSD1中发现了无义突变p.R450*，该突变存在于所有9个IA家族成员中。此外，我们在其他IA家系中发现了同一基因的7个潜在突变。我们观察到，在斑马鱼或小鼠中敲除Thsd1会导致自发性脑出血和过早死亡，这表明THSD1在维持脑血管完整性方面发挥了作用。THSD1编码一种分子功能未知的蛋白质。值得注意的是，THSD1在内皮细胞中表达，内皮细胞是大脑动脉中重要的细胞层。有趣的是，IA病理学的一个特征是完整的内皮细胞的丢失，这表明内皮功能障碍在IA的形成中起着作用。THSD1已被证明与talin结合，talin是一种局部黏附蛋白，有助于细胞与细胞外基质的附着。研究还表明，当内皮细胞从基质中分离出来时，细胞会经历程序性细胞死亡。由于Thsd1缺乏导致出血，我们推测THSD1通过促进内皮细胞与基底膜的黏附而参与维持内皮细胞的完整性，从而介导细胞存活。在这一应用中，我们建议使用两种体外模型系统：1)从野生型和Thsd1缺失小鼠分离的血管内皮细胞和平滑肌细胞；2)从野生型受试者和THSD1 p.R450*突变的实际患者中诱导多能干细胞(IPSC)来源的血管内皮细胞和平滑肌细胞。通过免疫组织化学、免疫印迹和功能分析，我们的目标是确定THSD1是否定位于局灶性粘连，以及THSD1的破坏是否会导致局灶性粘连组装、与细胞外基质的粘连和细胞存活的改变。 异常表型的成功证明将提供对THSD1介导的IA形成机制的洞察，并有可能确定预防性治疗的分子靶点。