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

• 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
• 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

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 镰状细胞病(SCD)影响着大约10万美国人和全球数百万人，美国的医疗费用超过10亿美元 每年用于治疗频繁和不可预测的血管闭塞发作(VOE)。尽管同样，单基因疾病会导致 血红蛋白S(HBs)的产生导致成熟红细胞(RBC)在缺氧后呈镰刀状，VOES的频率 在SCD患者中的分布具有高度的变异性。镰状网织红细胞(未成熟红细胞)通过参与一系列 由细胞表面黏附分子介导的黏附事件，这些黏附分子延迟小血管中的血流，从而促进镰状和 红细胞在微血管中的包裹。羟基脲(HU)是SCD的主要治疗方法，减少但不能消除VOES， 因此，接受治疗的患者仍然有相当大的风险使VOE变得虚弱。最初，HU被用来诱导血红蛋白F(HBF) 表达具有抗病作用，尽管HU也通过增加一氧化氮(NO)和cGMP提供直接的临床益处 水平(NO第二信使)，减少黏附受体表达和红细胞与内皮的相互作用。有一些引人入胜的 有证据表明，HU通过上调依赖NO/cGMP的途径降低黏附受体活性来调节黏附； 然而，具体机制尚不清楚。更好地理解HU减少粘合剂相互作用的机制将揭示 新的治疗靶点，以有效减少SCD的VOES。我的长期目标是识别细胞和分子靶点，以帮助 开发有效的治疗方法以改善对SCD患者的护理。这个项目将提高我们对病原生物学的认识。 支持SCD的机制，并将使我们获得对影响临床表现的分子途径的新见解 和SCD的严重程度。研究提案中提出的新概念与详细的培训计划和来自 基础科学、翻译和临床研究人员组成的高度成功的团队也将促进我的职业发展。 极晚期抗原-4(VLA-4)是SCD中特征性最强的黏附受体，在网织红细胞和白细胞上高度表达 血细胞(WBC)。与其他整合素一样，VLA-4在功能上受细胞信号通路的调节，从而调节活性和结合 在SCD微环境中，对各种配体的亲和力提高。表达VLA-4的网织红细胞和WBC增加 在VOES期间和有严重疾病的SCD患者中，表型减少，但对HU治疗有反应。利用我们的 标准化的流式黏附生物测定，我们先前表明VLA-4结合可用于临床对SCD患者进行分层 根据疾病的严重程度并预测即将到来的VOES。我们还表明，网织红细胞和HBF水平与 在患者报告的VOES期间，VLA-4结合和VLA-4介导的黏附增加，而在HU治疗的SCD患者中减少。 最近，来自我的实验室的初步数据表明，HU降低了镰状网织红细胞中VLA-4的活性/结合亲和力。 其他研究表明，VLA-4的结合是通过一种依赖于激酶的机制来增强的，而通过NO/cGMP途径来减少 信号；然而，镰状红细胞的具体机制尚不清楚。我的中心假设是HU减少了RBC-内皮细胞 通过依赖于NO/cGMP的途径降低激酶活性，从而在SCD中相互作用。使用我们的标准化流道粘附力 通过实验、优化的流式细胞术和质谱学方法，我将在以下具体目标上验证这一假设： 1)确定HU对VLA-4活性/结合亲和力的影响；2)阐明HU调节VLA-4蛋白的机制。 镰状红细胞中的蛋白质相互作用和翻译后修饰。