Elevated Reactive Oxygen Species Production In Sickle Erythrocytes Is Modulated by a Pathway Involving Endothelin-1, TGFβ1, PKC, and Rac GTPases
Authors: George Alex , Koochaki Sebastian , Pushkaran Suvarnamala , Mohandas Narla , Zheng Yi , Joiner Clinton H. , Kalfa Theodosia A 
Published: October 2019

Abstract
Abstract 1634
Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease. One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle erythrocytes. Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. The potential role of enzymatic mechanisms in ROS production in RBCs has not been fully explored. One candidate for enzyme-mediated ROS production in SS RBCs is NADPH oxidase, which is activated by the small Rho GTPases Rac1 and Rac2 in a variety of cell types (Hordijk P.L. Circ. Res. 2006; 98:453-462).
Using flow cytometry with 5-(and 6-)-chloromethyl-2`,7`-dichlorodihydrofluorescein diacetate, a peroxide-sensitive probe, we determined that ROS generation is elevated in HbSS RBCs by 150–250% relative to that in HbAA RBCs. The NADPH oxidase NOX subunit homologs NOX1 and NOX5 were expressed in erythrocytes, and treatment of SS RBCs with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) reduced ROS generation in a dose-dependent manner. Inhibition of PKC or Rac activity by the small molecule-inhibitors calphostin and NSC23766, respectively, also resulted in decreased ROS production in HbSS-RBCs in a dose-dependent fashion, while PKC activation by phorbol 12-myristate 13-acetate (PMA) increased ROS production. This effect of PMA could be inhibited by parallel treatment with NSC23766, implicating a PKC-Rac axis in erythrocyte ROS production. Enzymatic ROS production in sickle RBCs was dependent on the availability of free intracellular calcium, since it was inhibited by BAPTA-AM, a cell-permeable calcium chelator. Moreover, we found that ROS generation in SS RBCs was modulated by humoral factors. Incubation of AA RBCs in blood type-matched SS patient plasma resulted in increased ROS generation, while the incubation of SS RBCs in AA plasma decreased ROS production. Immunoblotting of AA and SS RBC membranes with specific antibodies revealed receptors for the inflammatory signaling molecules TGFβ1 and Endothelin-1 (ET-1), both of which are present in elevated levels in the plasma of patients with SCD. Incubation of AA RBCs with TGFβ1 or ET-1 resulted in increased Rac activation and increased ROS production in these treated cells.
Our results suggest that ROS production in sickle RBCs is mediated by NADPH oxidase through Ca2+-regulated PKC and Rac signals, which in turn are modulated by plasma TGFβ1 and ET-1 via their receptors. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte deformation and fragility in sickle cell disease. Erythrocyte ROS generation, RBC lysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive feedback loop to drive the pathophysiology of sickle cell disease. These data may offer new therapeutic targets to counteract inflammation and RBC fragility and deformation in sickle cell disease.

Disclosures:
No relevant conflicts of interest to declare.