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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。 MPN 其特征是红细胞、血小板和/或白细胞产生过多。血栓形成风险 MPN 被认为主要是继发于过量的克隆性 MPN 细胞。但目前来看，互动血管内皮细胞和克隆性 MPN 细胞之间的关系尚不清楚。克隆 MPN 增长受到推动由 Janus 激酶信号转导子和转录激活子 (JAK-STAT) 信号传导失调引起。这 JAK2V617F+突变发生在高达70%的MPN患者中，使血栓形成的风险增加6倍。此外，MPN 患者在慢流内脏脉管系统中发生 VTE 的风险较高。一些体外和体内研究表明，具有 JAK2V617F+ 突变的内皮细胞 (EC) 表达亲粘附和血栓形成蛋白，表明 EC 信号传导可能导致血栓形成增加。我的小学目的是确定 EC 激活如何导致 MPN 血栓形成。我的中心假设是在 EC 血管中，JAK2V617F+ 突变会引发促炎和血栓级联反应。在初步在研究中，我评估了从 JAK2V617F+ 患者分离的血液生长内皮细胞 (BOEC)。在 JAK2V617F+ 中 BOEC 和 TNF-α 激活的 JAK2WT EC 中，ruxolitinib 和 fedratinib（JAK1/2 抑制剂已批准用于 MPN) 降低组织因子 (TF) 的表达和活性。此外，与 JAK2WT EC 相比，JAK2V617F+ BOEC 表达较高水平的血管性血友病因子 (VWF) 和生长停滞特异性 6 (Gas6) 蛋白。气体6 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与受体 Axl、MERTK 和 Tyro3 结合后单核细胞激活。有趣的是，在初步研究中， JAK2V617F+ 个体的 Gas6、Axl 和 MERTK 血浆水平显着高于对照组。重要的是，最近的研究表明，阻断 Gas6-Axl 通路会杀死 JAK2V617F+ 造血干细胞体外细胞，缩小了 JAK2V617F+ 小鼠的脾脏大小并延长了生存期。然而，这些研究确实不评估 Gas6-Axl-MERTK 轴是否导致 MPN 血栓形成。表型变异限制使用 JAK2V617F 动物模型来评估止血和血栓形成。因此，我建议使用内皮化微流体模型来评估 JAK2V617F 表达如何增加 EC 激活。使用内皮化微流体模型，我将在生理剪切下培养 JAK2V617F+ EC 以评估变化具有促凝和粘合功能。此外，我将评估促粘附和血栓形成的相互作用 JAK2V617F+ EC 和全血之间。我还将探讨 JAK2V617F+ EC 中的 Gas6-Axl-MERTK 信号传导如何增加促凝和促粘附环境。总的来说，拟议的研究将建立剪切对 JAK2V617F+ EC 激活的贡献并评估 JAK2V617F+ 中的 Gas6-Axl-MERTK 信号传导促血栓激活。