Mn Porphyrin-Based Redox Active Drugs Improve Anemia and Reduce Organ Damage in a Murine Model of Sickle Cell Disease
Authors: Thamilarasan Madhan , Estupinan Rodolfo , Zennadi Rahima 
Published: November 2019

Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene that leads to devastating downstream consequences including chronic hemolytic anemia, episodic vascular occlusion, and cumulative oxidative organ damage resulting in death. Targeting the mechanisms leading to organ injury could bring out therapeutic approaches. Sickle red blood cell (RBC) oxidative damage, which leads to hemolysis, could participate in endothelial activation and organ damage. Recently, we show that NADPH oxidases (NOXs) are a major player in generating reactive oxygen species (ROS) within sickle RBCs to mediate adhesion and vaso-occlusion. We have now evaluated the effects of reducing ROS in sickle RBCs on hemolysis, and its consequences on endothelial activation and organ damage in sickle mice in vivo. To inactive RBC NOXs, thus reduce ROS levels, we used the redox-active manganese (Mn) porphyrins, MnTnBuOE-2-PyP5+ (MnBuOE) and MnTE-2-PyP5+ (MnE), commonly known as superoxide dismutase (SOD) mimics. Treatments of sickle mice for 4 weeks with 0.1 mg/kg MnBuOE and 1 mg/kg MnE significantly reduced sickle RBC ROS production profile compared to sickle RBC ROS levels in the control vehicle group (p<0.05), suggesting that Mn porphyrins protects sickle RBCs especially against NOX activation. Mn porphyrins also significantly affected apoptosis-like RBC death called eryptosis characterized by accelerated membrane senescence and PS externalization. Sickle mice-treated with vehicle showed 4.98±0.6% annexin V-positive sickle RBCs. However, 0.1 mg/kg MnBuOE and 1 mg/kg MnE markedly decreased the percentage of annexin V-binding sickle RBCs by 54% (p=0.0009) and 56% (p=0.0007), respectively. As a result of improved eryptosis, RBC counts rose from 5.23 x 106/mL in vehicle-treated sickle mice to 5.99 x 106/mL in MnE-treated mice. Hemoglobin (Hb) levels also rose from 7 g/dL in the vehicle group to 8.55 g/dL in MnE-treated mice. Accordingly, hematocrits rose from 26.1% in vehicle-treated mice to 28.59% in MnE-treated mice. Yet, reticulocyte counts were not significantly different between vehicle and MnE-treated sickle mice, implying less hemolysis and not an increase in RBC production. The effect of reduced hemolysis by Mn porphyrins on oxidative stress in different organs was next assessed. MnBuOE and MnE decreased ROS levels in the kidneys (p<0.03 for MnBuOE and MnE), liver (p<0.05 for MnBuOE and MnE), and spleen (p<0.004 for MnBuOE and MnE) compared to the vehicle-treated group. Since organ ROS levels were lowered, this outcome may be accompanied by down-regulation of activation of endothelial adhesion molecules VCAM-1, ICAM-1, and P-selectin, markers of endothelial dysfunction and disease severity and/or RBC and leukocyte adhesion in SCD. Indeed, adhesion molecule expression assays by qRT-PCR showed that MnBuOE suppressed ICAM-1 and VCAM-1 expression in the lungs (p<0.0001 and p=0.0054, respectively) and kidneys (p=0.0072 and p=0.0004, respectively), and P-selectin expression (p=0.0058) in the kidneys. MnE also down-regulated the expression of ICAM-1 in the lungs (p<0.0001) and kidneys (p=0.0125), and VCAM-1 (p=0.0038) in the kidneys. These drugs had no effect on ICAM-1, VCAM-1, and P-selectin expression in the liver and spleen. Based on these data, we assumed that Mn porphyrins might rescue damage of organs typically affected and impaired in SCD. Apoptosis and necrosis of the kidneys, liver, and spleen were assessed using annexin V apoptosis detection kit and H&E staining. Sickle mice treated with vehicle showed marked apoptosis in the kidney, liver, and spleen (p=0.0314) tissues, and necrosis. However, 0.1 mg/kg MnBuOE and 1 mg/kg MnE significantly decreased apoptosis (p<0.0001) and necrosis in the kidneys, and liver (p=0.0355). MnBuOE also reduced apoptosis (p=0.0314) and necrosis in the spleen. These data suggest that Mn porphyrins can alleviate organ impairment and damage via at least RBC and endothelial oxidative stress reduction. As a result of these beneficial outcomes of reduced hemolysis, endothelial oxidative damage and dysfunction, Kaplan-Meier survival curves showed a dramatic prolonged survival of sickle mice challenged with TNFα (p = .0009, log-rank test). Our data suggest that Mn porphyrins, by repressing NOX-mediated hemolysis could represent a novel therapeutic intervention to especially alleviate hemolysis-mediated endothelial activation and organ damage in SCD.

Disclosures
No relevant conflicts of interest to declare.