The Role of THSD1 in Endothelial Cell Adhesion and Survival

描述（由申请人提供）：颅内动脉瘤破裂（IA）是蛛网膜下腔出血（SAH）的常见原因，是导致过早死亡的主要原因。虽然环境因素被认为在IAs的形成和破裂中起作用，但家族聚集性强烈表明遗传成分。10%至20%的患者有IAs和动脉瘤性SAH的阳性家族史，与一般人群相比，一级亲属患病风险增加了5倍。人类基因组中的许多区域都与家族性IA风险有关。然而，致病的基因变异尚未被确定。我们的长期目标是确定突变时使个体易患IA的基因，确定导致IA疾病的分子途径，并制定阻止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，我们发现了编码血栓反应蛋白1型结构域蛋白1的THSD1无义突变p.R450*，该突变存在于IA的所有9个家族成员中。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，在斑马鱼或小鼠中敲低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在形成脑动脉重要细胞层的内皮细胞中表达。有趣的是，IA病理的一个标志是完整内皮的丧失，这表明内皮功能障碍在IA形成中的作用。THSD1已被证明与talin结合，talin是一种协助细胞附着到细胞外基质的黏附蛋白。研究还表明，当内皮细胞脱离底层基质时，细胞会发生程序性细胞死亡。由于Thsd1缺乏导致出血，我们假设Thsd1通过促进内皮细胞与基底膜的粘附参与维持内皮的完整性，从而介导细胞存活。在这项应用中，我们建议使用两种体外模型系统：1)从野生型和thsd1缺失小鼠中分离的血管内皮细胞和平滑肌细胞；2)诱导多能干细胞（iPSC）来源的血管内皮细胞和平滑肌细胞，来自野生型受试者和THSD1 p.R450*突变的实际患者。通过免疫组织化学、免疫印迹和功能分析，我们旨在确定THSD1是否定位于局灶黏附，以及THSD1的破坏是否会导致局灶黏附组装、对细胞外基质的粘附和细胞存活的改变。

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.