Identifying Mechanisms Involved in Hydroxyurea-Mediated Reduction in Vaso-occlusive Adhesive Events in Sickle Cell Disease

确定羟基脲介导的镰状细胞病血管闭塞性粘附事件减少机制

基本信息

批准号：
10724590
负责人：
Jennell White
金额：
$ 15.16万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-05 至 2028-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10724590
关键词：
Adhesions Adhesives Affect Affinity Affinity Chromatography Alternative Therapies American Antibodies Area Area Under Curve Basic Science Binding Biological Assay Biological Availability Blood Vessels Blood flow Blood specimen CD34 gene Caring Cell Adhesion Cell Adhesion Molecules Cell surface Cells Clinical Communication Coupled Cyclic GMP Cytolysis Data Development Effectiveness Endothelial Cells Erythroblasts Erythrocytes Erythroid Event Fetal Hemoglobin Flow Cytometry Fluorescence Frequencies Goals Harvest Health Care Costs Hematopoietic stem cells Hemoglobinopathies Immunoprecipitation In Vitro Integrin alpha4beta1 Integrins Knowledge Leadership Leukocytes Ligands Liquid Chromatography Mass Spectrum Analysis Measures Mediating Mendelian disorder Mentored Research Scientist Development Award Mentors Mentorship Methods Molecular Molecular Conformation Molecular Target Monoclonal Antibodies Nitric Oxide Outcome Pathway interactions Patient Care Patients Pharmaceutical Preparations Phosphorylation Phosphotransferases Post-Translational Protein Processing Production Proteins Proteome Protocols documentation Recombinant Proteins Reporting Research Personnel Research Proposals Reticulocytes Risk Role Sampling Series Severities Severity of illness Sickle Cell Sickle Cell Anemia Sickle Hemoglobin Signal Pathway Signal Transduction Standardization TFRC gene Testing Training Trypsin Up-Regulation Vascular Cell Adhesion Molecule-1 Vascular Endothelial Cell Whole Blood adhesion receptor career career development cellular targeting disease phenotype effective therapy experience hydroxyurea improved indexing insight magnetic beads new therapeutic target novel protein protein interaction receptor expression receptor function response sickling skills standard of care symptomatic improvement tandem mass spectrometry

项目摘要

Project Summary Sickle cell disease (SCD) affects ~100,000 Americans and millions worldwide, with healthcare costs in the U.S. exceeding $1 billion annually to treat frequent and unpredictable vaso-occlusive episodes (VOEs). Despite the same, monogenic disease that results in the production of hemoglobin S (HbS) causing mature red blood cells (RBCs) to sickle following deoxygenation, the frequency of VOEs amongst SCD patients is highly variable. Sickle reticulocytes (immature RBCs) contribute to VOEs by participating in a series of adhesive events mediated by cell surface adhesion molecules that delay blood flow in small blood vessels which promote sickling and entrapment of RBCs in the microvasculature. Hydroxyurea (HU), the mainstay therapy for SCD, reduces but does not eliminate VOEs, hence treated patients remain at considerable risk for debilitating VOEs. Initially, HU was administered to induce hemoglobin F (HbF) expression with anti-sickling effects, although HU also provides immediate clinical benefits by increasing nitric oxide (NO) and cGMP levels (NO 2nd messenger) and decreasing adhesion receptor expression and RBC-endothelial interactions. There is some compelling evidence that HU modulates adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity; however, specific mechanisms are unclear. A better understanding of HU mechanisms that reduce adhesive interactions will reveal novel therapeutic targets to reduce VOEs effectively in SCD. My long-term goal is to identify cellular and molecular targets to aid in the development of effective therapies to improve the care for SCD patients. This project will enhance our knowledge of the pathobiological mechanisms underpinning SCD and will allow us to gain new insights into molecular pathways that influence the clinical manifestations and severity of SCD. Novel concepts proposed in the research proposal combined with a detailed training plan and mentorship from a highly accomplished team of basic science, translational, and clinical researchers will also facilitate my career development. Very late antigen-4 (VLA-4), the best characterized adhesion receptor in SCD, is highly expressed on reticulocytes and white blood cells (WBCs). Like other integrins, VLA-4 is functionally regulated by cell signaling pathways to modulate activity and binding affinity to a wide variety of ligands elevated in the SCD micro-environment. VLA-4 expressing reticulocytes and WBCs are elevated during VOEs and in SCD patients with severe disease phenotypes, but decreased in response to HU therapy. Utilizing our standardized, flow adhesion bioassay, we previously showed that VLA-4 binding can be used clinically to stratify SCD patients based on disease severity and predict impending VOEs. We have also shown that reticulocyte and HbF levels strongly correlate with VLA-4 binding and VLA-4-mediated adhesion is increased during patient-reported VOEs and decreased in HU-treated SCD patients. More recently, preliminary data from my lab demonstrate that HU reduces VLA-4 activity/binding affinity in sickle reticulocytes. Others have shown that VLA-4 binding is enhanced by a kinase-dependent mechanism and reduced through NO/cGMP pathway signaling; yet, specific mechanisms in sickle RBCs are unclear. My central hypothesis is that HU reduces RBC-endothelial interactions in SCD by decreasing kinase activity through a NO/cGMP-dependent pathway. Using our standardized flow adhesion assay, optimized flow cytometry protocol, and mass spectrometric approach, I will test this hypothesis in the following specific aims: 1) Determine the effect of HU on VLA-4 activity/binding affinity; and 2) Elucidate HU mechanisms that modulate VLA-4 protein- protein interactions and post-translational modifications in sickle RBCs.

项目摘要镰状细胞疾病（SCD）在全球范围内影响约100,000美国人和数百万美元，美国的医疗保健费用超过10亿美元每年处理频繁且不可预测的血管熟悉发作（VOES）。尽管如此，单基因疾病导致血红蛋白S（HBS）的产生，导致成熟的红细胞（RBC）在脱氧后，VOES的频率在SCD患者中，有高度可变。镰状网状细胞（未成熟的RBC）通过参加一系列由细胞表面粘附分子介导的粘附事件，这些分子延迟了小血管中血液流动的促进，促进疾病和 RBC在微脉管系统中的夹带。羟基脲（HU），SCD的主要疗法，减少但不会消除Voes，因此，经过治疗的患者仍然处于使人衰弱的风险。最初，施用HU诱导血红蛋白F（HBF）具有防水作用的表达，尽管HU还通过增加一氧化氮（NO）和CGMP提供了立即的临床益处水平（没有第二信使）和粘附受体表达和RBC-内层相互作用。有一些引人注目的 HU通过上调NO/CGMP依赖性途径以降低粘附受体活性来调节粘附的证据。但是，具体机制尚不清楚。对减少粘合剂相互作用的HU机制有更好的理解将揭示在SCD中有效减少VOE的新型治疗靶标。我的长期目标是确定细胞和分子靶标，以帮助开发有效的疗法来改善SCD患者的护理。该项目将增强我们对病理学的了解 SCD支撑的机制，将使我们能够对影响临床表现的分子途径获得新的见解和SCD的严重程度。研究提案中提出的新颖概念结合了一个详细的培训计划和指导基础科学，转化和临床研究人员的高度成就团队也将促进我的职业发展。抗原4（VLA-4）是SCD中最佳特征的粘附受体，在网状细胞和白色上高度表达血细胞（WBC）。像其他整合素一样，VLA-4在功能上受细胞信号传导途径调节，以调节活性和结合 SCD微环境中升高的各种配体的亲和力。 VLA-4表达网状细胞和WBC升高在VOE和患有严重疾病表型的SCD患者中，但对HU治疗的响应减少。利用我们的标准化的流程粘附生物测定，我们先前表明VLA-4结合可在临床上用于分层SCD患者基于疾病的严重程度，并预测即将来临的VOE。我们还表明，网状细胞和HBF水平与在患者报告的VOE期间，VLA-4结合和VLA-4介导的粘附量增加，并且在HU治疗的SCD患者中减少。最近，我实验室的初步数据表明，HU降低了镰状网状细胞中的VLA-4活性/结合亲和力。其他人则表明，VLA-4结合通过激酶依赖性机制增强，并通过NO/CGMP途径降低信号传导；然而，镰状RBC中的特定机制尚不清楚。我的中心假设是HU减少了RBC-内皮通过通过NO/CGMP依赖性途径降低激酶活性，在SCD中的相互作用。使用我们的标准化流程粘附测定，优化的流式细胞仪方案和质谱方法，我将在以下特定目的中检验这一假设： 1）确定HU对VLA-4活性/结合亲和力的影响； 2）阐明调节VLA-4蛋白质的HU机制镰状RBC中的蛋白质相互作用和翻译后修饰。