Regulation of Endothelin-1-Mediated Reactive Oxygen Species Production by Protein Disulfide Isomerase in Endothelial Cells: Role in Sickle Cell Disease

Abstract 1073

Endothelial cell activation and increased levels of endothelin-1 (ET-1) have been proposed as important contributors to vaso-occlusion in the pathophysiology of Sickle Cell Disease (SCD). We have reported a role for ET-1 receptor antagonists in improving hematological complications of two transgenic mouse models of SCD in vivo (Rivera A., 2008, Amer J Physiol). Activation of endothelial cells leads to, among other factors, increased levels of reactive oxygen species (ROS) and protein disulfide isomerase (PDI). PDI mediates redox modifications, catalyzes disulfide interchange reactions in the plasma membrane and is up-regulated under hypoxic conditions as commonly observed in SCD. We tested the hypothesis that ET-1 would regulate PDI activity in endothelial cells. We now report on the detection of PDI and ET-1 receptor B by western blot analyses in membranes from the human endothelial cell line, EA.hy926 (EA). We studied the effects of ET-1 on cellular ROS production by fluorimetry of DCFDA-AM loaded EA cells and observed that 10⁻⁷ M ET-1 for 60 mins led to significant increases in cellular ROS production (1720±78 vs 1447±86 Relative Fluorescent Units (RFU), P<0.001, n=6) that were significantly blocked by ET-1 receptor B antagonist, 10⁻⁶ M BQ-788 (1720±78 vs 1426±55 RFU, P<0.0007, n=3). We also tested the effects of the specific ET-1 receptor B agonist, 10⁻⁷ M BQ-3020 on cellular ROS production and observed similar results (1748±194 vs 1447±86 RFU, P<0.02, n=3). We then studied the effect of a well-described PDI inhibitor, monoclonal antibodies to PDI (RL90), on ET-1 stimulated ROS production and showed that RL90 likewise blocked ET-1 stimulated ROS production in EA cells (P<0.001, n=3), thus suggesting a role for PDI in ET-1-stimulated ROS production. To further explore the role of PDI on ROS production, we employed a molecular approach and show that siRNA against PDI likewise led to reduced ET-1-stimulated ROS production (P<0.0001, n=2) that was associated with significantly reduced PDI mRNA levels in siRNA transfected cells, but not when cells were transfected with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and GAPDH and β2 microglobulin as endogenous controls. We then optimized conditions to measure PDI activity using fluorescently labeled GSSG conversion to GSH in supernatants of ET-1-stimulated cells. We observed that incubation of EA cells for 60 mins with 10⁻⁷ M ET-1 was associated with increased extracellular PDI activity (232±183 to 664±94 (RFU), n=3, P<0.02) that was blunted by PDI siRNA knockdown (1731±147 to 757±141 RFU, n=2) when compared to scrambled siRNA transfected cells. Consistent with these data we observed increases in PDI protein levels in supernatants of EA cells stimulated with ET-1 by ELISA. Moreover, incubation of EA cells for 12 hr with 10⁻⁸ M ET-1 led to increases in cell-associated PDI levels by western blot analyses. We then tested the in vivo effects of ET receptor antagonist on plasma PDI activity in the BERK mouse model of SCD. We blocked ET-1 receptors in vivo by treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed a reduction of plasma PDI activity when compared to vehicle treatment (67.7±3 to 34.3±6, RFU, P<0.03, n=3). We also characterized the effects of ET-1 on PDI expression in EA cells, using quantitative RT-PCR with ABI TaqMan probes and GAPDH and β-actin as endogenous controls and observed that stimulation of EA cells with 10⁻⁸ M ET-1 for 4 hr was associated with increased PDI mRNA expression levels that were 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Thus, our results provide evidence to suggest that endothelin-1 receptor blockade leads to improved measures of ROS via regulation of PDI and thus implicate PDI as a novel regulator of Sickle Cell Disease pathophysiology.