Single-Stranded Oligonucleotide Binding to P-Selectin Inhibits Adhesion of Sickle Red Blood Cells and Leukocytes to Endothelial Cells in Sickle Cell Model Mice: Novel Therapeutics for Vaso-Occlusive Episodes.

Abstract 1545

Poster Board I-568

To date, a variety of therapeutic approaches to sickle cell disease (SCD) have been explored, however, knowledge about the efficacy of anti-adhesive agents in this disorder is limited. In SCD, P-selectin expressed on endothelial cells plays a key role in leukocyte recruitment as well as in the adhesion of sickle red blood cells (sRBC) to the endothelium. The interaction of P-selectin and its ligands is thus likely to contribute to impairment of the microvascular flow and thereby to the development of painful vaso-occlusive episodes. Studies with anti-P-selectin antibody and P-selectin-deficient mice support the notion that P-selectin-directed interventions may be a potential approach to the treatment of vaso-occlusive episodes in SCD. Aptamers, short single-stranded oligonucleotides, have been developed for a wide range of therapeutic targets (Keefe & Schaub, 2008). Aptamers bind molecular targets with high affinity and specificity, do not elicit immune responses, and can be readily delivered to the vascular compartment intravenously or subcutaneously. Although anti-P-selectin aptamers have been shown to inhibit leukocyte rolling in vitro and to display efficacy in mouse models for inflammation, the anti-adhesion activity of anti-P-selectin aptamers has never been evaluated in SCD. The purpose of this study was to determine in vivo whether the anti-P-selectin aptamer ARC5690 can inhibit adhesion of sickle RBC and leukocytes to vascular endothelial cells in the bone marrow microvasculature of SCD model mice. Knockout-transgenic SCD mice generated by Ryan et al. (Ryan et al., 1997) were used in this study. SCD mice were injected with 20 mg/kg ARC5690, scrambled aptamer ARC5694 (negative control), or vehicle (saline). Anti-P-selectin monoclonal antibody was also used as a positive control. After 2.5 hours, the mice were subjected to 1 hour of hypoxia (12% O₂ in air) followed by 1 hour of reoxygenation at room air. Intravital observations of the bone marrow microcirculation were performed to monitor adhesive interactions between blood cells and endothelial cells. Leukocytes were labeled in vivo with rat anti mouse CD45 antibody conjugated with PE which were infused via the carotid artery. Sickle RBC were obtained from donor mice, fluorescently labeled in vitro with 2,7-bis-(carboxyethyl)-5-(and-6) carboxyfluorescein, and infused through the carotid artery. Effects of ARC5690 on sRBC and leukocyte adhesive interactions were compared to those of ARC5694 and vehicle. Administration of the anti-P-selectin aptamer ARC5690 significantly reduced sRBC adhesion (p<0.05) in SCD mice compared to those treated with vehicle. The anti-adhesive activity of ARC5690 for sRBC was comparable to anti-P-selectin monoclonal antibody, whereas the scrambled aptamer did not show any anti-adhesive activity. We also studied the effects of the anti-P-selectin-specific ARC5690 on leukocyte flow dynamics. Mice pretreated with ARC5690, but not those treated with scrambled aptamer ARC5694, showed a greater than four-fold decrease in leukocyte rolling (p<0.001) and a six-fold decrease in adhesion (p<0.001) compared with vehicle-treated mice. Again, the anti-adhesive activity of ARC5690 and its effect on leukocyte rolling and adhesion were similar to those of the anti-P-selectin antibody. Furthermore, ARC5690 improved blood circulation compared to vehicle-treated mice as shown by increased RBC velocity and d wall shear rates. In conclusion, our study demonstrates significant anti-adhesive activities of ARC5690, which reduces the adhesion of sRBC and leukocytes to the vascular endothelium in SCD model mice. ARC5690 may represent a novel therapeutic strategy that can be used to treat vaso-occlusive episodes in SCD