Basic Investigation and Translational Applications Concerning the Cell and Molecular Biology of Blood and Vascular Cells

本申请中建议的研究涵盖并扩展了校长的长期利益 研究血液和血管细胞的细胞和分子生物学。建议的计划包括 两个雄心勃勃、有影响力的项目，旨在填补各自领域的重要概念空白， 长期以来一直被认为与NHLBI的总体任务有关；即(1) PECAM-1在血管细胞功能中的作用；(2)新生儿同种免疫性血小板减少症的病理生理学 (NAIT)。PECAM-1(也称为CD31)是一种细胞黏附和信号受体，在循环中发挥作用 血细胞限制细胞激活的速度和程度。PECAM-1也是表达最高的 内皮细胞-细胞连接的组成部分，在那里它起着同嗜性粘附性应激反应的作用 维持内皮细胞连接完整性和加速血管通透性屏障恢复的蛋白质 在炎症性或血栓形成之后。我们将利用我们最近对三维 PECAM-1亲和结合结构域的结构，我们创新的PECAM-1靶向工具的开发 和动物模型，以及我们的发现，PECAM-1受到构象亲和力调节的影响 PECAM-1作为治疗广泛血管通透性的新靶点的潜力 疾病，包括败血症引起的血管渗漏和缺血再灌注损伤。该计划还将 专注于开发新的工具、模型和治疗方法来治疗NAIT-一种罕见但具有灾难性临床重要性的疾病 由父系遗传抗原产生的母体抗体引起的出血性疾病 在重新穿过胎盘并与胎儿和/或新生儿血小板结合的胎儿血小板上，导致 血小板减少症通常严重到需要输血，在最严重的情况下会导致颅内 出血和宫内死亡。尽管在治疗方面取得了进展，但NAIT仍然是导致 足月婴儿的颅内出血，通常导致终生残疾。我们建议将最近的 CRISPR基因编辑技术产生巨核细胞的变革性进展 祖细胞、巨核细胞和血小板从诱导的多能干细胞中建立转化 诊断平台旨在缩小现有的“诊断差距”，以改善NAIT的治疗和护理-- 即建立能够长期自我更新、冷冻保存、 和分布；从而提供潜在取之不尽的用于诊断的iPS来源的血小板， 研究用途(以及未来可能的治疗用途)。CRISPR技术还使我们能够开发出一种新的 NAIT的人源化小鼠模型将使我们能够解决一系列悬而未决的血小板问题 同种异体豁免。综上所述，这项研究计划将应用尖端技术奠定基础 在这些疾病和相关的血液和血管疾病的诊断和治疗方面继续取得进展 与国家心肺和血液研究所的使命相关。

Studies proposed in this application encompass and extend long-standing interests of the Principal Investigator in the cell and molecular biology of blood and vascular cells. The proposed Program is comprised of two ambitious, impactful projects designed to fill important conceptual gaps in their respective fields, and that have long been judged to be of interest and relevance to the overall mission of the NHLBI; namely (1) The role of PECAM-1 in vascular cell function, and (2) The pathophysiology of neonatal alloimmune thrombocytopenia (NAIT). PECAM-1 (also known as CD31) is a cellular adhesion and signaling receptor that functions in circulating blood cells to limit the rate and extent of cellular activation. PECAM-1 is also the most highly-expressed component of the endothelial cell-cell junction, where it functions as a homophilic adhesive stress-response protein to maintain endothelial cell junctional integrity and speed restoration of the vascular permeability barrier following inflammatory or thrombotic challenge. We will exploit our recent determination of the three-dimensional structure of the PECAM-1 homophilic binding domain, our development of innovative PECAM-1-targeted tools and animal models, and our discovery that PECAM-1 is subject to conformational affinity modulation to examine the potential for PECAM-1 to serve as a novel therapeutic target for a wide range of vascular permeability disorders, including sepsis-induced vascular leakage and ischemia-reperfusion injury. This Program will also focus on developing new tools, models and treatments for NAIT – a rare, but catastrophic, clinically important bleeding disorder caused by maternal antibodies generated in response to paternally-inherited antigens present on fetal platelets that re-cross the placenta and bind to fetal and/or neonatal platelets, resulting in thrombocytopenia often serious enough to require transfusion, and in the most severe cases causing intracranial hemorrhage and intrauterine death. Despite advances in treatment, NAIT remains the leading cause of intracranial hemorrhage in full-term infants, often leading to lifelong disability. We propose to combine recent transformative advances in CRISPR gene editing technology with the ability to generate megakaryocyte progenitor cells, megakaryocytes, and platelets from induced pluripotent stem cells to establish a transformative diagnostic platform designed to narrow the existing “diagnostic gap” to improve treatment and care of NAIT - namely the creation of platelet alloantigen-specific cell lines capable of long-term self-renewal, cryopreservation, and distribution; thereby providing a potentially inexhaustible source of iPS-derived platelets for diagnostic, investigative, (and potentially future therapeutic) use. CRISPR technology has also allowed us to develop a novel humanized mouse model of NAIT that will allow us to resolve a series of outstanding issues in platelet alloimmunity. Taken together, this research program will apply cutting-edge technology to lay the groundwork for continuing advances in the diagnosis and treatment of these and related blood and vascular disorders of relevance to the mission of the National Heart, Lung and Blood Institute.