Shear-Mediated Erythrocyte Nitric Oxide Production Is Differentially Regulated in Patients with Sickle Cell Disease
Authors: Detterich Jon A , Xu Iris , Suriany Silvie , Ponce Derek , Bush Adam M , Wood John C , Meiselman Herbert J , Forman Henry Jay , Coates Thomas D. 
Published: October 2019

Abstract
Introduction: Sickle cell disease (SCD) is an inherited hemoglobinopathy in which hemoglobin polymerizes when deoxygenated, causing increased red blood cell (RBC) stiffness and RBC sickling. This leads to microcirculatory slowing and occlusion, in turn causing vaso-occlusive crises acutely and microcirculatory damage chronically. RBC are known to signal blood vessels in the microcirculation, regulating vessel tone. Nitric oxide (NO) is a potent vasodilator and key regulator of vascular smooth muscle tone throughout the vasculature. However, the possibility that RBC make NO and that this might contribute to vascular tone, particularly in arterioles is controversial. Modulation of RBC NO production due to deformation of RBC by shear stress could have important physiological consequences in SCD where RBC deformability is decreased and dependent on the polymerization state of hemoglobin S.
Objective: To determine if shear stress induced deformation induces change in NO production in individual RBC and if the magnitude and time course of this NO differs between RBC from patients with sickle cell disease and normal controls.
Methods: NO production was measured using imaging microscopy of fluorescently labeled RBC exposed to shear after being allowed to adhere to a flow chamber attached to the microscope stage. Four SCD patients on simple chronic transfusion therapy, 4 SCD patients not on chronic transfusion therapy and 4 healthy control subjects were recruited from the hematology clinics at Children's Hospital Los Angeles under an IRB approved protocol. RBC underwent histopaque separation then washed 3x and resuspended in KRP buffer. The RBC were then incubated with 4 micromolar DAF-FM Diacetate (4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate, ThermoFisher Scientific), a fluorescent indicator related to NO production. RBC were then exposed to three experimental conditions: 1. buffer alone; 2. 1 milimolar arginine (substrate for NOS NO production); 3. 1 milimolar arginine and 1 milimolar L-NAME (a non-specific NOS inhibitor). The RBC were placed in poly L-lysine coated microfluidic chambers and placed under no shear conditions for 30 minutesk, then sheared at 0.5Pa for 30minutes. Individual cell analysis was performed using Image J analysis software (FIJI).
Results: There is a slow baseline rise of NO fluorescent signal without exposure to shear and there is a large, acute increase in NO fluorescent signal with exposure to shear stress. (figure 1) The baseline fluorescent rate of rise is increased in non-transfused sickle cell subjects (p<0.0001) compared to transfused SCD patients and healthy control subjects, suggesting higher basal rate of NO production. (figure 2) We found a significant increase in fluorescence in response to shear in all three groups, with the transfused SCD patients showing a higher rate of change and higher magnitude fluorescence response. (figure 1) Addition of arginine increased the shear induced NO production in all three groups (P<0.05). The baseline NO related fluorescence was blunted with the addition of L-NAME in healthy subjects, whereas it was unchanged in both the transfused and non-transfused SCD groups. There was no significant change in shear-mediated NO related DAF fluorescence after L-NAME was added.
Discussion: The results here indicate that RBC produce NO upon shear and the basal rate of NO production is higher in non-transfused patients with sickle cell disease. Addition of arginine increases both the magnitude and rate of NO production in all three groups. SCD patients on chronic transfusion therapy demonstrate a larger increase in NO response to shear. The increased response of NO related fluorescence with the addition of arginine suggests NOS has a role in shear mediated RBC NO production; however, L-NAME blockade does not significantly blunt the response in the presence of arginine. Shear induced RBC NO production may play an important role in regulation of microvascular profusion and would likely be modulated by the RBC deformability differences due to HbS polymerization.
Conclusion: There is basal and shear dependent NO production in both AA and SCD RBC, and RBC NOS is differentially regulated in SCD and AA RBC.
Figure 1 Response of RBC NO related fluorescence to shear stress Figure 1. Response of RBC NO related fluorescence to shear stress Figure 2 Basal rate of NO related DAF fluorescence. Figure 2. Basal rate of NO related DAF fluorescence.

Disclosures
Wood: Ionis Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy; Vifor: Consultancy; Vifor: Consultancy; World Care Clinical: Consultancy; World Care Clinical: Consultancy; Celgene: Consultancy; Celgene: Consultancy; AMAG: Consultancy; AMAG: Consultancy; Apopharma: Consultancy; Apopharma: Consultancy; Biomed Informatics: Consultancy; Biomed Informatics: Consultancy.