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

项目摘要 镰状细胞病（SCD）影响约10万美国人和全球数百万人，美国的医疗保健费用超过10亿美元 治疗频繁和不可预测的血管闭塞发作（VOE）。尽管同样的单基因疾病会导致 血红蛋白S（HbS）的产生导致成熟红细胞（RBC）在脱氧后镰刀形，VOE的频率 在SCD患者中是高度可变的。镰状网织红细胞（未成熟的红细胞）通过参与一系列 由细胞表面粘附分子介导的粘附事件，其延迟小血管中的血流，从而促进镰状化， 红细胞被困在微血管中。羟基脲（HU）是SCD的主要治疗方法，可减少但不能消除VOE， 因此，接受治疗的患者仍有相当大的使人衰弱的VOE风险。最初，给予HU以诱导血红蛋白F（HbF） 尽管HU也通过增加一氧化氮（NO）和cGMP的表达提供了直接的临床益处， 水平（NO第二信使）和降低粘附受体表达和RBC-内皮相互作用。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; 然而，具体机制尚不清楚。更好地理解HU机制，减少粘附相互作用将揭示 有效降低SCD中VOE的新治疗靶点。我的长期目标是确定细胞和分子靶点， 开发有效的治疗方法以改善SCD患者的护理。该项目将提高我们对病理生物学的认识， 支持SCD的机制，并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概念，结合详细的培训计划和来自 一个由基础科学、转化和临床研究人员组成的高水平团队也将促进我的职业发展。 极晚期抗原-4（VLA-4）是SCD中最具特征的粘附受体，在网织红细胞和白色红细胞上高度表达， 血细胞（WBC）。与其他整合素一样，VLA-4在功能上受细胞信号传导途径调节，以调节活性和结合 对SCD微环境中升高的多种配体的亲和力。表达VLA-4的网织红细胞和WBC升高 在VOE期间和具有严重疾病表型的SCD患者中，但对HU治疗的反应降低。利用我们 标准化，流动粘附生物测定，我们以前表明，VLA-4结合可用于临床分层SCD患者 根据疾病的严重程度和预测即将发生的VOE。我们还表明，网织红细胞和HbF水平强烈相关， VLA-4结合和VLA-4介导的粘附在患者报告的VOE期间增加，在HU治疗的SCD患者中减少。 最近，来自我实验室的初步数据表明，HU降低了镰状网织红细胞中的VLA-4活性/结合亲和力。 其他研究表明，VLA-4结合通过激酶依赖性机制增强，通过NO/cGMP途径减少 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RBC-内皮细胞 通过NO/cGMP依赖性途径降低激酶活性，从而抑制SCD中的相互作用。使用我们的标准化流动粘附 分析、优化的流式细胞术方案和质谱方法，我将在以下具体目标中检验这一假设： 1)确定HU对VLA-4活性/结合亲和力的影响;和2）阐明调节VLA-4蛋白的HU机制。 镰状红细胞中的蛋白质相互作用和翻译后修饰。

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.