Effect of Voxelotor on Red Blood Cell Adhesion Under Normoxia Using an Endothelialized Microfluidic System

Introduction: Sickle cell disease (SCD) is characterized by the polymerization of hemoglobin S (HbS) that results in sickle red blood cell (sRBC) formation under deoxygenation. Subsequent alterations in the rheological properties of sRBC lead to abnormal deformability, adhesion, and ultimately vasculopathy. Vascular occlusion leads to repeated cycles of organ ischemia and pain. Voxelotor (Oxbryta, Global Blood Therapeutics, South San Francisco, CA, USA) is an FDA-approved medication that reversibly binds to sickle hemoglobin in a dose-dependent fashion to increase its oxygen affinity and reduce sickling and polymerization. Previous studies using voxelotor under hypoxic conditions have shown an improvement in blood flow (Azul & Wood, 2020) and sRBC deformability (Dufu et al., 2018). As previously published, endothelialized microfluidic devices have been used to quantify sRBC adhesion (Kucukal et al., 2021). This experimental study is the first in-vitro assessment of the effect of voxelotor on adhesion under normoxic conditions using an endothelialized microfluidic system.

Methods: Human umbilical vein endothelial cells (HUVECs, Lonza, Morristown, USA) were cultured within a microfluidic system for at least 72 hours prior to experiments. HUVECs underwent four-hour activation with tumor necrosis factor alpha (TNFα) at a concentration of 20ng/mL. Under an IRB-approved study, whole blood samples from 12 patients with HbSS SCD were collected in EDTA tubes and red blood cells were isolated via centrifugation. RBCs were then resuspended in basal media (Lonza) with HEPES for pH-adjustment and treated with and without voxelotor (Selleckchem, Radnor, PA, USA) to a final hematocrit of 20% and concentration of 600μM. After one-hour in the incu-shaker, the samples were injected through the activated channels under normoxia at a rate of 2μL/min. Non-adherent cells were then removed by flushing the channels with additional media with HEPES. Adhered cells were counted via Adobe Photoshop Software. Statistical significance was calculated via paired t-test.

Results: RBC adhesion ranged between 580 and 15,492 cells in untreated, activated samples and between 516 and 14,216 cells in voxelotor-treated, activated samples (Figure 1). Voxelotor treatment resulted in decreased sRBC adhesion in all but two of twelve samples and the percentage of adhesion reduction ranged between 7 and 70 percent (Figure 2, p=0.2). No conclusions were made when comparing patient's laboratory values (white blood cell, hemoglobin, platelets, absolute neutrophil count, absolute reticulocyte count, lactate dehydrogenase) to percent of adhesion reduction.

Conclusion: Our results show that voxelotor does result in adhesion reduction under normoxia although, overall, the drug did not significantly decrease RBC adhesion. We theorize that by improving oxygen affinity and decreasing polymerization, voxelotor may limit the downstream effects of polymerization, specifically adhesion and vaso-occlusion. One major limitation is that RBCs were not deoxygenated and as mentioned above, voxelotor has improved RBC rheology under hypoxia. Therefore, future experiments will assess the effect of voxelotor on adhesion under hypoxic conditions.

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