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

本申请中提出的研究涵盖并扩展了委托人的长期利益 血液和血管细胞的细胞和分子生物学研究者。拟议方案包括 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，人们一直认为这与NHLBI的总体使命有关，即：（1）作用 PECAM-1在血管细胞功能中的作用，以及（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CD 31）是一种细胞粘附和信号传导受体，在循环中起作用。 血细胞来限制细胞活化的速率和程度。PECAM-1也是最高表达的 内皮细胞-细胞连接的组成部分，在那里它起着嗜同性粘附应激反应的作用 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 在炎症或血栓形成挑战后。我们将利用我们最近确定的三维 结构的PECAM-1嗜同性结合域，我们的创新PECAM-1靶向工具的发展 和动物模型，我们发现PECAM-1受到构象亲和力调节，以检查 PECAM-1作为广泛血管通透性的新治疗靶点的潜力 疾病，包括脓毒症诱导的血管渗漏和缺血-再灌注损伤。该方案还将 专注于开发新的工具，模型和治疗NAIT -一种罕见的，但灾难性的，临床上重要的 由母体抗体引起的出血性疾病，该抗体是对存在的父系遗传抗原的应答产生的 对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，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， 出血和宫内死亡。尽管在治疗方面取得了进展，NAIT仍然是导致 足月婴儿颅内出血，往往导致终身残疾。我们建议将最近的联合收割机 CRISPR基因编辑技术的革命性进展，能够产生巨核细胞 祖细胞，巨核细胞和血小板从诱导多能干细胞，以建立一个转化 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- 即产生能够长期自我更新，冷冻保存， 和分布;从而提供了一种潜在的取之不尽的来源于iPS的血小板用于诊断， 研究（和潜在的未来治疗）用途。CRISPR技术也使我们能够开发出一种新的 NAIT的人源化小鼠模型，这将使我们能够解决一系列悬而未决的问题，在血小板 同种免疫综合来看，这项研究计划将应用尖端技术， 在诊断和治疗这些和相关的血液和血管疾病的不断进步， 与国家心肺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.