Hypoxia-Inducible Factors and Neutrophil Heterogeneity in Myeloproliferative Neoplasm-Associated Venous Thrombosis

项目摘要/摘要 这个K08职业发展奖详细介绍了一个为期5年的培训计划，以推进我成为 一位R01资助的内科科学家专注于阐明骨髓增生性疾病的血栓形成机制 为了寻找新的治疗靶点，改善对MPN患者的护理。在颁奖期间 在此期间，我将继续发展对血栓形成的机理理解和模型方面的专业知识，获取 新的科学技能，先进的翻译能力，并产生出版物和数据，将作为 为成功申请R01奠定基础。在我的主要导师Rafal Pawlinski博士的指导下， 共同导师乔纳森·塞罗迪博士和艾莉森·莫利特诺博士将通过联合行动来实现这些培训目标 讲授课程的工作，参加系列研讨会，研究经验，以及我的顾问的指导 委员会审议阶段。奈杰尔·基博士和约瑟夫·普拉查尔博士将担任该提案的顾问。 这项科学提案旨在确定JAK2V617F阳性MPN的血栓形成机制。血栓形成 仍然是主要MPN亚型真性红细胞增多症和特发性红细胞增多症的主要死亡原因 血小板增多症。MPN患者循环血细胞既有数量增多，又有质的异常。 目前的治疗方案旨在使循环血细胞数量正常化，以改善血液流变学。 然而，尽管有目前的治疗方法，25%的MPN患者在确诊后会经历血栓事件。 了解血栓形成的基础并制定一级和二级预防策略是关键 这一领域未得到满足的需求。Prchal博士和我最近报告说，缺氧诱导因子(HIF)的上调- MPN患者中性粒细胞介导性基因表达与血栓病史的关系。重要的是，我们 还发现中性粒细胞组织因子(Tf)的基因表达增加，组织因子是外源性疾病的主要启动者 凝血途径。我们扩展了这一发现，证明了MPN中性粒细胞具有转铁蛋白 促凝血剂活性。纤溶酶原激活物抑制物-1(PAI-1)是纤溶的主要生理性抑制物； 在MPN患者中，其基因表达水平也明显升高。因此，我假设JAK2V617F驱动的增长 HIF活性降低导致TF和PAI-1表达上调，从而增加MPN血栓形成的风险。在目标1中， 我将使用静脉血栓形成(VT)的小鼠模型来评估(1)增加 JAK2V617F转基因复制到室性心动过速，(2)抑制缺氧诱导因子-1α或缺氧诱导因子-2α对室性心动过速的影响，(3) JAK2V617F突变在造血细胞和内皮细胞中对室速的影响，以及(4)中性粒细胞转铁蛋白对室速的影响。在……里面 目的研究0、1、2个JAK2V617F突变等位基因对中性粒细胞基因异质性的影响 单细胞RNA测序重叠单细胞基因分型在MPN患者中的表达。两个目标 是互补但独立的，并将确定预防MPN血栓形成的新靶点和SET 这是进一步调查的舞台。

PROJECT SUMMARY/ABSTRACT This K08 Career Development Award details a 5-year training program to advance my career goal of becoming an R01-funded physician-scientist focused on elucidating thrombotic mechanisms in myeloproliferative neoplasms (MPN) in order to find new treatment targets and improve care of MPN patients. During the award period, I will continue developing expertise in mechanistic understanding of and models of thrombosis, acquire new scientific skills, advance translational capabilities, and generate publications and data that will serve as the foundation for a successful R01 application. Under the guidance of my primary mentor, Dr. Rafal Pawlinski, and co-mentors, Drs. Jonathan Serody and Alison Moliterno, these training objectives will be met by a combination of didactic course work, participation in seminar series, research experience, and mentoring by my advisory committee. Dr. Nigel Key and Dr. Josef Prchal will serve as advisors on the proposal. The scientific proposal is aimed at identifying thrombotic mechanisms in JAK2V617F-positive MPN. Thrombosis remains the leading cause of mortality in the major MPN subtypes polycythemia vera and essential thrombocythemia. Circulating blood cells are both increased in quantity and qualitatively abnormal in MPN. Current treatment paradigms aim to normalize the number of circulating blood cells to improve blood rheology. However, despite current therapies 25% of MPN patients will experience a thrombotic event after diagnosis. Understanding the basis of thrombosis and developing primary and secondary prevention strategies are key unmet needs in this field. Dr. Prchal and I recently reported that upregulation of hypoxia-inducible factor (HIF)- mediated gene expression in neutrophils of MPN patients associated with thrombosis history. Importantly, we also found increased neutrophil gene expression of tissue factor (TF), the primary initiator of the extrinsic pathway of coagulation. We extended this finding, demonstrating that MPN neutrophils possess TF procoagulant activity. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiologic inhibitor of fibrinolysis; its gene expression was also increased in MPN patients. I therefore hypothesize that JAK2V617F-driven increases of HIF activity leads to upregulation of TF and PAI-1, thereby increasing the risk of MPN thrombosis. In Aim 1, I will use a mouse model of venous thrombosis (VT) to evaluate (1) the functional consequence of increasing JAK2V617F transgene copies to VT, (2) the effect of HIF-1α or HIF-2α inhibition to VT, (3) the contribution of JAK2V617F mutation in hematopoietic versus endothelial cells to VT, and (4) the effect of neutrophil TF to VT. In Aim 2, I will evaluate the effect of 0, 1, or 2 JAK2V617F-mutated alleles on heterogeneity of neutrophil gene expression in MPN patients using single cell RNA sequencing with overlayed single cell genotyping. The 2 Aims are complementary but independent and will identify new targets for the prevention of MPN thrombosis and set the stage for additional investigations.