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

点击翻译按钮获取中文摘要

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.