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（由申请人提供）：颅内动脉瘤（IA）破裂是蛛网膜下腔出血（SAH）的常见原因，是过早死亡的主要原因。虽然环境因素被认为在IA的形成和破裂中发挥作用，但家族聚集强烈表明遗传成分。10%至20%的患者有IA和蛛网膜下腔出血的阳性家族史，与一般人群相比，一级亲属发生疾病的风险增加了5倍。人类基因组中的许多区域与家族性IA风险有关。然而，致病的遗传变异尚未确定。我们的长期目标是确定基因突变时易患IA的个体，确定导致IA疾病的分子途径，并制定治疗策略，以阻止IA的增长。使用全基因组连锁和外显子组分析一个大的家庭，我们确定了无义突变p.R450* THSD 1编码凝血酶敏感蛋白1型结构域的蛋白1，是目前在所有9个家庭成员与IA。此外，我们在其他IA家族中发现了7个相同基因的潜在突变。我们观察到，敲低斑马鱼或小鼠的Thsd 1会导致自发性脑出血和过早死亡，这表明THSD 1在维持脑血管完整性方面的作用。THSD 1编码一种分子功能未知的蛋白质。值得注意的是，THSD 1在内皮细胞中表达，内皮细胞形成脑动脉中的重要细胞层。有趣的是，IA病理学的一个标志是完整内皮的丧失，这表明内皮功能障碍在IA形成中发挥着作用。THSD 1已被证明结合塔林蛋白，一种有助于细胞附着到细胞外基质的粘着斑蛋白。也已经表明，当内皮细胞从下面的基质分离时，细胞经历程序性细胞死亡。由于Thsd 1缺乏导致出血，我们假设THSD 1通过促进内皮细胞粘附于基底膜，从而介导细胞存活，参与维持内皮的完整性。在本申请中，我们提出使用两种体外模型系统：1）从野生型和Thsd 1缺失小鼠分离的血管内皮和平滑肌细胞;和2）来自野生型受试者和具有THSD 1 p.R450* 突变的实际患者的诱导多能干细胞（iPSC）衍生的血管内皮和平滑肌细胞。使用免疫组织化学，免疫印迹和功能测定，我们的目标是确定THSD 1是否定位于粘着斑，如果THSD 1的破坏将导致改变粘着斑组装，粘附细胞外基质和细胞存活。 异常表型的成功证明将提供对THSD 1介导的IA形成机制的深入了解，并可能确定预防性治疗的分子靶点。