Platelet TSP1 Mediates Vascular Disease and pulmonary hypertension in Sickle Cell

描述（由申请人提供）：这是一份针对Enrico Novelli博士的K23奖申请，他是匹兹堡大学成人镰状细胞性贫血项目主任、血液学家、医学系终身职位助理教授。Novelli博士是良性血液学领域的学术领袖，专注于镰状细胞病和止血的转化研究。Novelli博士之前在血液学方面拥有丰富的研究经验，并因其在SCD方面的研究成功获得了研究奖项。他组建了一个由导师和顾问组成的多学科团队，以指导他的职业生涯走向独立，并协助完成本申请中提出的研究。这项资助将有助达致以下职业目标：(1)发展实验室方法方面的专业知识，而这些方法对在SCD进行转化研究至关重要；(2)进一步深入了解临床试验设计和人类疾病调查的生物统计学基础知识；(3)成为SCD血管生物学方面的专家。他计划通过完成由匹兹堡大学赞助的临床研究理学硕士学位来补充他的研究活动，参加关于拨款写作和负责任的研究行为的预定培训活动，并出席他感兴趣领域的国际会议。在K23资助期结束时，他将有几篇同行评审的第一作者出版物和大量数据，这些数据将构成RO1或其他同等资助的核心。本应用程序的重点是SCD的血管生物学。在SCD中，突变的血红蛋白S在缺氧时聚合，驱动红细胞（RBC）依赖性血管闭塞和溶血。这些过程导致血小板和止血激活、肺动脉高压和血管疾病。转基因敲除镰刀（BERK）小鼠只表达人类¿-和？s -珠蛋白通过显示NO生物利用度降低、NO介导的血管反应性受损和肺动脉高压来模拟人类SCD。最近，血小板颗粒蛋白血小板反应蛋白-1 （TSP1）被发现通过与细胞表面受体CD47结合，有效抑制生理性NO信号传导。Novelli博士的初步数据表明，SCD患者血浆中循环TSP1水平升高。支持这一建议的临床前研究也表明，特异性的TSP1-CD47配体受体相互作用不仅抑制内皮细胞中的NO信号，而且增加了NO信号

DESCRIPTION (provided by applicant): This is an application for a K23 award for Dr. Enrico Novelli, a Hematologist, Assistant Professor in the Tenure Stream in the Department of Medicine and Director of the University of Pittsburgh Adult Sickle Cell Anemia Program. Dr. Novelli is establishing himself as an academic leader in the field of benign hematology with a specific focus on translational research in sickle cell disease and hemostasis. Dr. Novelli has prior extensive research experiences in hematology and has been successful in obtaining research awards for his studies on SCD. He has assembled a multidisciplinary team of mentors and advisors to guide his career towards independence and to assist with the completion of the research proposed in this application. This grant will be critical to achieve the following career objectives: (1) to develop expertise in laboratory methodologies crucial to the conduct of translational research in SCD; (2) to gain further in-depth knowledge of clinical trial design and biostatistics fundamental in the investigation of human disease; and (3) to become an expert in the vascular biology of SCD. He plans to complement his research activity with the completion of a Master of Science in Clinical Research sponsored by the University of Pittsburgh, with participation in scheduled training activities on grant writing and responsible conduct of research, and with attendance to international meetings in his areas of interest. By the end of the funding period of this K23 award, he will have several first-authored peer-reviewed publications and a critical mass of data that will form the core of an RO1 or other equivalent grant. The focus of this application is on the vascular biology of SCD. In SCD, mutant hemoglobin S polymerizes when deoxygenated, driving red blood cell (RBC)-dependent vaso-occlusion and hemolysis. These processes lead to platelet and hemostatic activation, pulmonary hypertension and vascular disease. Transgenic-knockout sickle (BERK) mice that express exclusively human ¿- and ?S-globins mimic SCD in humans by displaying reduced NO bioavailability, impaired NO-mediated vascular reactivity and pulmonary hypertension. Recently, the platelet ¿-granule protein thrombospondin-1 (TSP1) was found to potently inhibits physiologic NO signaling, via binding to the cell surface receptor CD47. Preliminary data from Dr. Novelli now demonstrate that circulating TSP1 levels are increased in the plasma of patients with SCD. Preclinical studies supporting this proposal also show that the specific TSP1-CD47 ligand receptor interaction not only inhibits NO signaling in endothelial cells, but also increases reactive oxygen species (ROS) production and endothelin A (ETA) receptor expression in smooth muscle, both canonical vasoconstrictive and mitogenic pathological signaling pathways contributing to pulmonary hypertension. Extensive preliminary data using chimera cross transplantation systems confirm that CD47 is critical in the development of PH in the transgenic sickle cell mouse. These findings inform the grant's overarching hypothesis that during platelet activation in patients with SCD, increases in circulating plasma levels of TSP1, via binding to the CD47 receptor, disrupt pulmonary vascular endothelial NO production and stimulate smooth muscle ROS production and endothelin 1 signaling, which lead to pulmonary hypertension in murine models and human subjects with SCD. This grant will also explore the hypothesis that therapeutic disruption of the TSP1-CD47 ligand-receptor interaction will both prevent and reverse pulmonary hypertension in SCD. This hypothesis will be tested via the following aims: (1) to examine for the first time whether plasma from human subjects with SCD TSP1 levels in the plasma of patients with SCD disrupt the vasodilator/vasoconstrictor balance in endothelium and smooth muscle by inhibition of NO signaling and increasing ROS and ETA levels; (2) to employ unique mutant murine models to determine the role of circulating TSP1 and CD47 in SCD-associated vasculopathy and pulmonary hypertension, and test the therapeutic efficacy of blocking TSP1-CD47 to prevent/mitigate SCD-based pulmonary hypertension; and (3) to test TSP1 levels in several large cohorts of patients with SCD and explore the correlation of TSP1 with platelet activation, pulmonary hypertension and vascular disease. This proposal is strengthened by the complementary and rich tool set used to investigate the hypothesis, the vast resources available within the Vascular Medicine Institute, a state of the art facility within the University of Pittsburgh devoted to the study of vascular biology, and captures the pioneering work of Dr. Novelli's mentors in defining hemolysis in the pathophysiology of SCD (Dr. Mark Gladwin's lab) and the TSP1-CD47 nexus as an upstream regulator of NO signaling (Dr. Jeff Isenberg's lab). Definition of this pathway will therefore present a novel therapeutic target for the vascular complications of SCD.