Endothelial-Specific Knockdown Of Caveolin-1 Lowers Circulating Protein Disulfide Isomerase Levels and Is Associated With Changes In Erythrocyte Volume Homeostasis

We have recently reported that Endothelin-1 (ET1), a potent vasoconstrictor peptide, is implicated in the pathophysiology of Sickle Cell Disease (SCD) via increased circulating Protein Disulfide Isomerase (PDI) activity (Prado, 2013 FASEB J). PDI is a multifunctional enzyme of the thioredoxin superfamily that mediates redox modifications, catalyzes disulfide interchange reactions in the plasma membrane, regulates KCNN4 channel and erythrocyte volume and is up-regulated under hypoxic conditions as commonly observed in SCD. In erythrocytes, ET1 stimulates PDI activity via activation of ET1 receptor B (ETRB). However, the precise mechanisms by which ET1 leads to increases in PDI are not entirely clear. There is evidence that activation of endothelial cells leads to increased PDI secretion and that ETRBs form a complex with caveolin-1 (CAV1) within caveolae to mediate ET1’s cellular effects. We tested the hypothesis that reduction of CAV1 would alter PDI secretion. We studied the in vivo effects of endothelial-specific CAV1 knockdown on circulating PDI activity in mice. We optimized conditions to measure circulating PDI using fluorescently labeled GSSG conversion to GSH. We now report that circulating plasma PDI levels were significantly decreased in CAV1 knockdown mice when compared to wild-type littermates (WT) (7.44±0.70 vs 10.93±2.66, n=7, P<0.05). In addition and consistent with our report showing a role for PDI in erythrocyte volume regulation, we also observed lower cell hemoglobin concentration mean (CHCM) and hemoglobin distribution width (HDW) that was associated with increased erythrocyte and reticulocyte mean cell volume (MCV) in blood from CAV1 knockdown mice when compared to WT (n=13 and n=19, respectively, P<0.005). We then isolated early cultures of mouse aortic endothelial cells (MAEC) from these mice and measured PDI activity following 24 hrs of incubation in 0.4% fetal bovine serum. Our results show that MAEC from CAV1 knockdown mice had lower PDI secretion when compared to cells from WT mice (99.4±16 vs 129.9±35, n=5, P<0.03). We then studied the effects of ET1 on PDI secretion from human endothelial cells. We detected PDI and ETRB by western blot analyses in membranes from the human endothelial cell line, EA.hy926 (EA). We observed that incubation of EA cells for 60 mins with 10^-7 M ET1 was associated with increased extracellular PDI activity (15.97±7.22 to 34.07±8.89 [RFU/mg protein], n=3, P<0.011) that was sensitive to preincubation with BQ788, a specific ETRB receptor antagonist (15.97±7.22 to 7.97±3.25 (RFU/mg protein), n=3, P<0.02). Similar increases in PDI were observed when cells were treated with the specific ETRB agonist, IRL1620 (1143±137 to 1593 207 RFU/mg protein). In addition, PDI siRNA knockdown was associated with reduced ET1-stimulated PDI activity when compared to scrambled siRNA transfected cells (1731±147 to 757±141 RFU, n=2). We then tested the effects of methyl-β-cyclodextrin to disrupt caveolae in these cells and observed a blunted IRL1620–stimulated PDI response (288±40 to 171±14 RFU/mg protein, n=3, P<0.025). We also characterized the effects of ET1 on PDI expression in EA cells, using quantitative RT-PCR with ABI TaqMan probes and β-actin as an endogenous control and observed that stimulation of EA cells with 10^-8 M ET1 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 for a heretofore unrecognized role of endothelial specific CAV1 in erythrocyte volume and circulating PDI levels. Supported by NIH R01HL090632 (AR) and R01HL104032 (LHP).