权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Engineered microsystems to assess endothelial contribution to myeloproliferative neoplasm thrombosis

工程微系统评估内皮对骨髓增生性肿瘤血栓形成的贡献

基本信息

批准号：
10674047
负责人：
Joan Denise Beckman
金额：
$ 17.12万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10674047
关键词：
Adhesions Adhesives Aftercare Animal Model Aspirin Autocrine Communication Automobile Driving Binding Biological Assay Blood Blood Cells Blood Platelets Cardiac Cell Separation Cells Cessation of life Data Devices Disease E-Selectin Endothelial Cells Endothelium Engineering Environment Erythrocytes Event Exhibits Family Flow Cytometry Future Genes Genetic Transcription Goals Growth Hematopoietic Hematopoietic stem cells Hemorrhage Hemostatic function Homologous Protein Immunofluorescence Immunologic In Vitro Individual Inflammatory Intercellular adhesion molecule 1 Interferon alpha JAK1 gene Janus kinase Lead Leukocyte Rolling Leukocytes MERTK gene Mediating Microfluidic Microchips Microfluidics Modeling Mononuclear Mus Mutation Myeloproliferative disease Operative Surgical Procedures Pathway interactions Patients Phenotype Physiological Plasma Platelet Activation Prevention strategy Production Protein S Proteins Research Risk Role STAT3 gene Secondary to Signal Pathway Signal Transduction Spleen Stretching TNF gene Techniques Testing Therapeutic Thromboplastin Thrombosis Training Transcription Coactivator Vascular Cell Adhesion Molecule-1 Vascular Endothelial Cell Vascular Endothelium Venous Venous blood sampling Western Blotting Whole Blood Work experience high risk hydroxyurea in vivo inhibitor microsystems monocyte mortality mouse model novel prevent receptor response shear stress targeted treatment thrombotic transcriptome sequencing venous thromboembolism von Willebrand Factor

项目摘要

PROJECT ABSTRACT Thrombosis is the leading cause of mortality among patients with myeloproliferative neoplasms (MPNs). MPNs are characterized by excessive production of red blood cells, platelets, and/or leukocytes. Thrombosis risk in MPNs is thought to be primarily secondary to excess clonal MPN cells. However, at present, the interaction between the vascular endothelium and clonal MPN cells is poorly characterized. Clonal MPN growth is driven by dysregulated Janus kinase-signal transductor and activator of transcription (JAK-STAT) signaling. The JAK2V617F+ mutation occurs in up to 70% of MPN patients and increases the risk of thrombosis 6-fold. Additionally, MPN patients have a higher risk of VTE in slow-flow splanchnic vasculature. Several in vitro and in vivo studies demonstrate that endothelial cells (EC) with the JAK2V617F+ mutation express pro-adhesive and thrombotic proteins, suggesting that EC signaling may contribute to increased thrombosis. My primary objective is to define how EC activation contributes to MPN thrombosis. My central hypothesis is that within the EC vascular, the JAK2V617F+ mutation evokes a pro-inflammatory and thrombotic cascade. In preliminary studies, I evaluated blood outgrowth endothelial cells (BOEC) isolated from JAK2V617F+ patients. In JAK2V617F+ BOECs and in TNF-α-activated JAK2WT ECs, ruxolitinib and fedratinib (JAK1/2 inhibitors approved for use in MPN) reduced tissue factor (TF) expression and activity. Additionally, Compared to JAK2WT ECs, JAK2V617F+ BOECs express higher levels von Willebrand factor (VWF), and growth arrest specific 6 (Gas6) protein. Gas6 is a vitamin-K dependent protein S homolog, which promotes both TF expression and triggers platelet and monocyte activation after binding to receptors Axl, MERTK, and Tyro3. Interestingly, in preliminary studies, JAK2V617F+ individuals had significantly higher plasma levels of Gas6, Axl, and MERTK than controls. Importantly, recent work has shown that blockade of the Gas6-Axl pathway kills JAK2V617F+ hematopoietic stem cells in vitro and reduces spleen size and prolongs survival in JAK2V617F+ mice. However, these studies did not evaluate whether the Gas6-Axl-MERTK axis contributes to MPN thrombosis. Phenotypic variability limits use of JAK2V617F animal models to assess hemostasis and thrombosis. Therefore, I propose to use endothelialized microfluidics models to assess how JAK2V617F expression increases EC activation. Using an endothelialized microfluidics model, I will culture JAK2V617F+ EC under physiologic shear to assess for changes in pro-coagulant and adhesive function. Furthermore, I will assess pro-adhesive and thrombotic interactions between JAK2V617F+ EC and whole blood. I will also explore how Gas6-Axl-MERTK signaling in JAK2V617F+ ECs increases the pro-coagulant and pro-adhesive environment. Collectively, the proposed research will establish the contribution of shear to JAK2V617F+ EC activation and evaluate Gas6-Axl-MERTK signaling in JAK2V617F+ pro-thrombotic activation.

项目摘要血栓形成是骨髓增生性肿瘤（MPN）患者死亡的主要原因。MPNs 其特征在于红细胞、血小板和/或白细胞的过度产生。血栓形成风险 MPN被认为主要继发于过量的克隆MPN细胞。目前，互动血管内皮细胞和克隆MPN细胞之间的差异很难被描述。克隆MPN增长是由 Janus激酶信号转导和转录激活因子（JAK-STAT）信号转导失调。的 JAK 2 V617 F+突变发生在高达70%的MPN患者中，并使血栓形成的风险增加6倍。此外，MPN患者在血流缓慢的内脏血管中发生VTE的风险更高。几项体外和体内研究表明，具有JAK 2 V617 F+突变的内皮细胞（EC）表达促粘附和血栓蛋白，表明EC信号可能有助于增加血栓形成。我的主要目的是确定EC激活如何促进MPN血栓形成。我的核心假设是，在EC血管中，JAK 2 V617 F+突变引起促炎和血栓级联反应。初步在研究中，我评估了从JAK 2 V617 F+患者分离的血液生长内皮细胞（BOEC）。在JAK 2 V617 F + 在BOEC和TNF-α激活的JAK 2 WT EC中，鲁索替尼和非德拉替尼（JAK 1/2抑制剂，批准用于 MPN）降低组织因子（TF）的表达和活性。此外，与JAK 2 WT EC相比，JAK 2 V617 F + BOEC表达更高水平的血管性血友病因子（VWF）和生长停滞特异性6（Gas 6）蛋白。Gas6 是一种维生素K依赖性蛋白S同系物，可促进TF表达并触发血小板和结合受体Axl、MERTK和Tyro 3后的单核细胞活化。有趣的是，在初步研究中， JAK 2 V617 F+个体具有比对照显著更高的Gas 6、Axl和MERTK血浆水平。重要的是，最近的工作表明Gas 6-Axl途径的阻断会杀死JAK 2 V617 F+造血干细胞在JAK 2 V617 F+小鼠中，在体外培养细胞并降低脾脏大小和脾脏存活率。然而，这些研究确实未评价Gas 6-Axl-MERTK轴是否有助于MPN血栓形成。表型变异限制使用JAK 2 V617 F动物模型评估止血和血栓形成。因此，我建议使用在内皮化微流体模型中，评估JAK 2 V617 F表达如何增加EC活化。使用在内皮化微流体模型中，我将在生理剪切下培养JAK 2 V617 F + EC以评估变化促凝血和粘附功能。此外，我将评估促粘附和血栓形成的相互作用 JAK 2 V617 F + EC和全血之间的差异。我还将探索JAK 2 V617 F + EC中的Gas 6-Axl-MERTK信号传导如何增加促凝血剂和促粘附剂环境。总的来说，拟议的研究将建立剪切对JAK 2 V617 F + EC活化贡献和评估JAK 2 V617 F+中的Gas 6-Axl-MERTK信号传导促血栓形成激活。