Inhibition of Protein Disulfide Isomerase Ameliorates Vaso-Occlusion and Thrombosis in a Mouse Model of Sickle Cell Disease

Introduction

Sickle cell disease (SCD) results from a single mutation in the β-globin gene. Altered erythrocyte physiology in SCD results in severe vascular complications, including vaso-occlusion (VOC) and painful crises along with various other manifestations, such as thrombosis and end organ damage. Extracellular protein disulfide isomerase (PDI) plays a critical role in the regulation of neutrophil recruitment, platelet activation and fibrin formation, processes implicated in the thrombo-inflammatory vasculopathy in SCD. We investigated the effect of a PDI inhibitor, isoquercetin, in combination with hydroxyurea, in TNFα-induced VOC and thrombosis in a mouse model of SCD. In addition, the effect of chronic PDI inhibition by isoquercetin on thrombo-inflammatory vasculopathy was also examined.

Methods

Humanized TOWNES knock-in mice, containing human normal (β^A/β^A, control) or sickle (β^S/β^S, SCD) hemoglobin β chains, were used as a mouse model of SCD. PDI inhibition in the plasma was achieved by oral gavage or daily diet feeding of isoquercetin. Intravital microscopy was performed to determine vaso-occlusive events in the venules and thrombus formation in the arterioles in TNFα-stimulated mice. Plasma levels of PDI and parameters for coagulation and vascular inflammation were determined by ELISA. Neutrophil accumulation in the organs was determined as the myeloperoxidase (MPO) levels in tissue homogenates.

Results

Compared to control mice, SCD mice have elevated plasma PDI levels. Gavage of isoquercetin resulted in reduced PDI activity in the plasma of treated mice (38±10% inhibition, p <0.05). Isoquercetin ingestion increased leukocyte rolling flux fraction in TNFα-treated SCD mice as monitored using intravital microscopy, whereas the influence on leukocyte adhesion and shear rate was minimal. When a reversal agent for PDI inhibitor, b'x (the isolated substrate binding domain of PDI) was co-infused, the effect of isoquercetin on leukocyte rolling was reversed, suggesting this effect of isoquercetin is mediated by PDI inhibition. We also determined the effect of isoquercetin in combination with hydroxyurea, a treatment with established efficacy in SCD, on the vaso-occlusive events in our model. We confirmed that hydroxyurea treatment elevated the plasma nitric oxide level as determined by plasma nitrite/nitrate content (11.5±1.9 μM vs 5.0±1.9 μM, p <0.05). Consistent with previous studies, hydroxyurea decreased leukocyte adhesion while the rolling flux fraction was not changed. However, combined treatment with isoquercetin and hydroxyurea in SCD mice increased the rolling flux fraction (15±3% vs 6±1%, p <0.05), decreased leukocyte adhesion (158±13 mm^-2 vs 289±24 mm^-2, p <0.05), and increased vessel shear rate (302±18 s^-1 vs 122±11 s^-1, p <0.05). We investigated in vivo thrombus formation in the arterioles of TNFα-treated SCD mice using intravital microscopy following laser injury. Compared to the control mice, SCD mice have significantly enhanced fibrin generation but normal platelet accumulation. Both isoquercetin and hydroxyurea inhibited TNFα-induced platelet accumulation (62±36% AUC-Area Under the Curve with isoquercetin and 48±17% AUC with hydroxyurea of untreated SCD) and fibrin generation (30±6% AUC with isoquercetin and 44±9% AUC with hydroxyurea of untreated SCD). To study the effect of chronic isoquercetin treatment in SCD, control and SCD mice were fed isoquercetin for 4 weeks. Daily consumption of isoquercetin resulted in reduction of plasma PDI activity (18±5% inhibition, p <0.05). Consistent with the intravital microscopic data, chronic PDI inhibition reduced neutrophil migration and accumulation into the lung, as determined by the MPO level (481±85 ng/mg vs 829±140 ng/mg, p <0.05) in the tissue homogenate. Chronic treatment with isoquercetin also reduced plasma fibrinogen degradation products (FDP) (102±15 μg/ml vs 183±25 μg/ml, p <0.05) in SCD mice. In addition, a correlation (Pearson r=0.651) was observed between reducing plasma FDP and lung MPO levels.

Conclusions

Acute and chronic PDI inhibition by isoquercetin may provide beneficial effect on vascular complications in a mouse model of SCD. PDI inhibition has the potential to ameliorate thrombo-inflammatory syndromes associated with SCD. These studies justify a longitudinal trial of PDI inhibitors in SCD.