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Roles of Red Blood Cell Derived Extracellular Vesicles in Complement Activation and Thromboinflammation in Sickle Cell Disease

红细胞衍生的细胞外囊泡在镰状细胞病补体激活和血栓炎症中的作用

基本信息

批准号：
10449795
负责人：
Ran An
金额：
$ 10.65万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
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 2023-02-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10449795
关键词：
Abnormal Red Blood Cell Acetylcysteine Adhesions Animal Model Autologous Award Biological Assay Biological Markers Biomedical Research Blood specimen CD2 gene Caring Cell Adhesion Cell surface Cells Characteristics Chemical Engineering Clinical Coagulation Process Complement Complement Activation Cytolysis Data Databases Deep Vein Thrombosis Deoxygenated Sickle Hemoglobin Deposition Diagnosis Disease Disease Outcome Drug Metabolic Detoxication Endothelial Cells Endothelium Enzyme-Linked Immunosorbent Assay Erythrocytes Evaluation Exhibits Exposure to GKLF protein Generations Heme Hemolysis Hemolytic-Uremic Syndrome Hemopexin Hypoxemia Hypoxia In Vitro Individual Inflammation Inflammatory Inpatients Knowledge Laboratories Lead Malaria Mediating Membrane Proteins Microfluidics Morbidity - disease rate Mus Mutation Outcome Outpatients Oxidative Stress Pain Parents Pathologic Pathway interactions Patients Periodicity Persons Phenotype Phosphatidylserines Physiological Plasma Play Polymers Population Heterogeneity Predisposition Process Prognosis Property Proteomics Recording of previous events Regulation Reverse Transcriptase Polymerase Chain Reaction Risk Role Sampling Scientist Sickle Cell Sickle Cell Anemia Sickle Hemoglobin Stress Surface Suspensions System Testing Therapeutic Intervention Thrombin Thrombosis Thrombus Transcriptional Regulation Transfusion Treatment Efficacy Western Blotting White Blood Cell Count procedure candidate selection comorbidity complement system emergency settings experience extracellular vesicles hydroxyurea improved individual patient mortality novel therapeutics patient population patient response polymerization pre-clinical assessment predict clinical outcome protein expression research clinical testing response screening shear stress sickling targeted treatment thromboinflammation treatment response vesicular release

项目摘要

PROJECT SUMMARY Sickle cell disease (SCD) is caused by abnormal polymerization of deoxygenated sickle hemoglobin, which damages red blood cells (RBCs), resulting in hemolysis and thromboinflammation. Sickled-RBCs lead to heterogeneous clinical complications in SCD and induce challenges in determining the exact therapeutic interventions. Extracellular vesicles (EVs) are released from cells, and can serve as vehicles for exchanging biomolecules and reflect the parent cell’s functional state. Complement system (CS) activation plays important roles in intravascular hemolysis, red blood cell EV (REV) generation, and thromboinflammation. In SCD, REVs express phosphatidylserine and transport heme that are capable of promoting thrombosis, microvascular inflammation triggering thrombus formation and cell adhesion. I hypothesize that in SCD: REVs will serve as a biomarker to predict CS activation and CS-mediated thromboinflammation, evaluate disease activity, and assist in preliminary evaluation of responses to therapeutic intervention. Here, I propose to use microfluidic systems and functional assays I developed to enhance the mechanistic understanding of stress-induced CS activation and REV generation, and the roles of REV in CS activation and thromboinflammation in heterogeneous population of patients with SCD. First, I will determine the responses of SCD RBC and complement system after exposure to individual and cumulated stresses including shear stress, hypoxia, and cycles of hypoxia- reoxygenation using microfluidic system. I will determine the role of stresses in CS activation, hemolysis, and REV generation. Autologous REVs generated under in vitro stresses and endogenous REVs isolated from plasma will be characterized for defining their surface protein expression, proteomic content, and effect on amplifying CS activation. I will refine the applied stresses to generate autologous REVs using samples at baseline states for mimicking endogenous REVs in samples at elevated disease state. Second, I will elucidate the roles of REV and CS activation in thromboinflammation. REV-mediated endothelial activation through Kruppel Like Factor 4 and complement activation will be assessed using both analytical and function endpoints including abnormal cellular adhesion and thrombus formation. Third, I will utilize analytical and functional assays to assess (using endogenous REVs) and predict (using autologous REVs) clinical outcome and efficacies of therapeutic intervention for individual patients. I will establish a temporal and progressive database of REV- initiated CS activation and thromboinflammation for assist patient-specific diagnosis and prognosis. This project will improve the understanding of the roles of CS and REV in hemolysis and thromboinflammation in SCD, and has the potential to establish a REV database to enable accurate evaluation of disease outcomes. Given my background as a chemical engineer, the K25 award will uniquely enable me to gain experience and become an independent scientist in basic and translational biomedical research.

项目总结镰状细胞病(SCD)是由脱氧镰状血红蛋白异常聚合引起的损害红细胞，导致溶血和血栓性炎症。镰刀状红细胞导致 SCD的不同临床并发症和在确定确切治疗方法方面的挑战干预措施。胞外囊泡是从细胞中释放出来的，可以作为交换的载体。并反映亲本细胞的功能状态。补体系统(CS)激活很重要在血管内溶血、红细胞EV(REV)生成和血栓性炎症中的作用。在SCD中，转速表达磷脂酰丝氨酸和运输血红素，能够促进血栓形成、微血管炎症引发血栓形成和细胞黏附。我假设在SCD：Revs将作为一个预测CS激活和CS介导的血栓炎症、评估疾病活动性和辅助的生物标记物在对治疗干预反应的初步评估中。在这里，我建议使用微流控系统和我开发的功能分析，以增强对应激诱导CS激活的机制的理解和REV的生成，以及REV在CS激活和异种血栓性炎症中的作用 SCD患者群体。首先，我将测定SCD红细胞和补体系统在治疗后的反应暴露在单个和累积的应力下，包括剪应力、低氧和循环低氧- 采用微流控系统进行复氧。我将确定应激在CS激活、溶血和 Rev世代。体外应激下产生的自体转速和分离的内源性转速将对血浆进行表征，以确定它们的表面蛋白表达、蛋白质组含量和对放大CS的激活。我将使用以下示例优化施加的应力以生成自体转速在疾病状态升高的样本中模拟内源性转速的基线状态。第二，我会澄清 REV和CS激活在血栓性炎症中的作用REV介导的内皮激活通过将使用分析和功能终点来评估Kruppel样因子4和补体激活包括异常细胞黏附和血栓形成。第三，我将利用分析和功能分析评估(使用内源性转速)和预测(使用自体转速)临床结果和疗效针对个别患者的治疗性干预。我会建立一个时间和进度数据库的牧师- 启动CS激活和血栓性炎症，以协助特定患者的诊断和预后。这个项目将提高对CS和REV在SCD的溶血和血栓炎症中的作用的理解，以及有可能建立REV数据库，以实现对疾病结果的准确评估。鉴于我的作为一名化学工程师，K25奖将使我获得独特的经验，并成为一名基础和转化型生物医学研究的独立科学家。