Assessment of Novel Erythroid Signaling Pathways Modulating Hydroxyurea-Mediated Reduction of Vaso-Occlusive Adhesive Interactions in Early Precursors of Sickle Red Blood Cells (RBCs)

Abstract

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). In addition to fetal hemoglobin induction, hydroxyurea (HU) reduces the frequency of VOEs by increasing nitric oxide (NO) bioavailability and decreasing adhesion receptor expression and RBC-endothelial interactions. Very late antigen-4 (VLA-4), the best characterized adhesion receptor in SCD, is highly expressed on circulating reticulocytes (immature RBCs) and white blood cells (WBCs) during VOEs and in SCD patients with severe disease phenotypes and, decreased in response to HU therapy. Like other integrins, VLA-4 is functionally regulated by cell signaling pathways to modulate activity and binding affinity to a wide variety of ligands that are elevated in the SCD micro-environment. There is highly intriguing evidence showing HU modulates adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity however specific mechanisms remain unclear. The objective of this study was to assess rapid cell signaling pathways involved in HU-mediated reduction in VLA-4 binding to endothelial vascular cell adhesion molecule-1 (VCAM-1). Whole blood (WB) was obtained from patients with SCD at benign clinic visits according to IRB-approved protocols. WB density-gradient centrifugation was used to isolate hematopoietic stem cells (HSCs) and WBCs from the buffy coat layer and, a CD71 monoclonal antibody (Ab) coupled with magnetic beads to isolate reticulocytes from the packed RBC layer. HSCs were matured to erythroblasts (EBs) in appropriate cell culture media and flow cytometric analyses assessed purity of blood samples using CD235a (exclusive WBC marker) and CD45 (exclusive RBC marker) Abs . Blood samples were pretreated with and without a therapeutically relevant hydroxyurea concentration (50mcM) followed by perfusion through micro-fluidic channels coated with VCAM-1 during pulsatile, shear flow (1.67Hz, 1.0 dyne/cm 2). Additionally, EBs were treated with HU combined and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1- one (ODQ; NO-dependent guanylate cyclase inhibitor). Adhered cells were quantified to establish an adhesion index. The non-parametric Wilcoxon matched pairs signed rank test was used to test the statistical differences between groups. Data are presented as mean ± standard error of mean. A p-value < 0.05 was considered statistically significant. There was a significant decrease in WB (p=0.01) and reticulocyte (p<0.0001) adhesion to VCAM-1 in HU-treated samples however, there was no effect on WBC (p>0.99) adhesion suggesting reticulocytes reduce the adhesive burden of blood cells in WB adhesion assays and modulation of VLA-4 binding differs in sickle RBCs when compared to WBCs. More recently, preliminary data from my lab demonstrated that EBs pretreated with HU reduces VLA-4 binding to VCAM-1 (from 126.5 ± 29.42 to 109.5 ± 29.09) by cGMP-dependent mechanisms (HU + ODQ; from 109.5 ± 29.09 to 120.2 ± 42.80). Laboratory-based static and flow adhesion assays have been used for decades to describe adhesion in SCD since earlier studies established that sickle RBCs are more adherent than non-sickle blood cells and, adhesion correlates with SCD severity. For example, the adhesion assay described above has been used to demonstrate that VLA-4 binding can stratify SCD patients based on disease severity and predict impending VOEs. Additionally, reticulocyte and HbF levels strongly correlate with VLA-4 binding to VCAM-1 and, VLA-4-mediated adhesion is increased during patient-reported VOEs and decreased in HU-treated SCD patients. This is the first SCD study demonstrating the role of HU in modulating VLA-4 binding to endothelial VCAM-1 through rapid erythroid signaling pathways. There is also compelling evidence involving downregulation of VLA-4 activity/binding affinity through NO/cGMP dependent mechanisms and enhancement of VLA-4 binding by kinase-dependent pathways. HU has also been shown to modulate adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity. Collectively, these data suggest that VLA-4 mechanisms may be targeted to therapeutically inhibit vaso-occlusive adhesive events that promote VOEs in SCD.