Regulation of Endothelin-1 Stimulated-Ca2+-Activated K+ Channel Activity by Protein Disulfide Isomerase Gene Knockdown in Endothelial Cells

Abstract Abstract 1080 Endothelial cell activation is as an important contributor to vaso-occlusion in the pathophysiology of Sickle Cell Disease. We have reported a role for dual endothelin-1 (ET-1) receptor antagonists in improving sickle cell pathophysiology in vivo via modulation of the Ca2+-activated K+ channel, KCNN4 (Rivera A., 2008, Amer J Physiol). Protein disulfide isomerase (PDI) catalyzes disulfide interchange reactions in the plasma membrane, mediates redox modifications and is up-regulated under hypoxic conditions. Thus KCNN4 maybe regulated by PDI in endothelial cells. We now report on the detection of KCNN4, 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 ET-1 on cytosolic Ca2+ levels by spectrofluorimetry of FURA-2AM loaded endothelial cells and observed that 10−9−10−7 M ET-1 led to dose-dependent increases in cytosolic Ca2+ levels that were blocked by pre-incubation with the ET-1 receptor B antagonist, BQ-788. We then studied the effects of 100 nM ET-1 on Ca2+-activated K+ channels and observed increased activity (153.7 vs. 187.0 mmol/L × h × mg protein) that was sensitive to clotrimazole (95.8 mmol/L × h × mg protein), a well-known inhibitor of KCNN4. We then studied the effects of two well-described PDI inhibitors, monoclonal antibodies to PDI and bacitracin, on channel activity and showed that these inhibitors significantly decreased channel activity in EA cells. Consistent with these observations, we show that siRNA against PDI likewise led to a reduction in channel activity (153.7 vs 29.5 mmol/L × h × mg protein, P<0.01, n=6) that was associated with significantly reduced PDI mRNA levels but not with scrambled siRNA as determined by quantitative RT-PCR with TaqMan detection probes and GAPDH and beta-2 microglobulin as endogenous controls. We then studied the effects of ET-1 on EA cells as well as in early cultures of mouse aortic endothelial cells (MAEC) using quantitative RT-PCR with TaqMan probes and GAPDH and beta-actin as endogenous controls and observed that stimulation of EA cells with 100 nM ET-1 for 4 hr was associated with increased mRNA expression of PDI levels that was 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Similar results were observed on PDI mRNA expression in MAEC incubated with 100 nM ET-1 for 4 hr. Furthermore, incubation of EA cells for 12 hr with 10 nM 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 in sickle transgenic BERK mice and observed a reduction of plasma PDI activity when compared to vehicle treatment (67.7±3 to 34.3±6, Relative Fluorescence Units, P<0.03, n=3). In these mice, we also observed a positive correlation in erythrocyte Gardos channel activity and PDI activity. Thus, our results implicate PDI as a novel regulator of KCNN4. We posit that regulation of PDI activity represents a novel target aimed at ameliorating complications associated with the pathophysiology of Sickle Cell Disease. Supported by NIH R01HL090632 to AR. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v120.21.1073.1073 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1073-1073

Author(s):

Patricia Neuman ◽

Gregory N. Prado ◽

Philip Asamoah ◽

Daphne Diaz ◽

Jose R Romero ◽

...

Keyword(s):

Endothelial Cells ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Ros Production ◽

Cell Disease ◽

Rt Pcr ◽

Endothelin 1 ◽

Endogenous Controls ◽

Protein Disulfide

Abstract 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. Disclosures: No relevant conflicts of interest to declare.

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Protein Disulfide Isomerase in Sickle Cell Disease

Blood ◽

10.1182/blood.v126.23.2166.2166 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2166-2166

Author(s):

Daniel Gil de Lamadrid ◽

Yaritza Inostroza-Nieves ◽

Joshua Cazares ◽

Isamar Alicea ◽

Analaura Santiago ◽

...

Keyword(s):

Endothelial Cells ◽

Mouse Models ◽

Sickle Cell ◽

Protein Disulfide Isomerase ◽

Channel Activity ◽

Cell Disease ◽

Disulfide Isomerase ◽

Gardos Channel ◽

Sickle Erythrocytes ◽

Protein Disulfide

Abstract Protein disulfide isomerase (PDI), a multifunctional oxidoreductase that regulates thrombus formation, leukocyte adherence to the endothelium and nitric oxide delivery, is present at high levels and regulates KCNN4 (Gardos Channel) activity in erythrocytes from humans with Sickle Cell Disease (SCD) and sickle mouse models. We reported evidence of elevated erythrocyte-surface associated and circulating PDI activity in humans and mouse models of SCD when compared to either wild-type mice, transgenic mice expressing normal human globins or otherwise healthy individuals; results that suggest a novel role for PDI in the pathophysiology of SCD through unclear mechanisms. We studied the effects of deoxygenation (5% O2) on PDI activity in endothelial cells and sickle erythrocytes from humans with SCD and two sickle transgenic knockout mouse models expressing human sickle hemoglobin, BERK and βSAntilles. Our data shows that deoxygenation of human and mouse sickle erythrocytes increased surface-associated reductive capacity that was sensitive to monoclonal antibodies against PDI (mAb PDI [RL-90]). We then studied sickle human erythrocytes and showed that PDI inhibitors (quercentin-3-rutinoside [Q3R] and mAb PDI) significantly reduced deoxy-stimulated sickle cell dehydration and Gardos channel activity (n=5; P <0.03). We characterized erythrocyte density with a phthalate density-distribution assay, generated density distribution curves and calculated the D50. Both mAb PDI and Q3R significantly reduced D50 when compared to vehicle (1.113±0.002 to 1.102±0.001 [P <0.0001]; and 1.101±0.002 [P <0.0001]; respectively). In contrast, incubation with exogenous PDI (3 nM) increased cellular dehydration (from D50= 1.110 ± 0.001 to D50= 1.115 ±0.001, P <0.01). We also measured the effect of hypoxia and endothelin-1 (ET-1; 10nM) in the human vascular endothelial cell line, EA.hy926. We observed that hypoxia induced PDI secretion that was further enhanced by co-incubation with ET-1 (10nM; n=3; P <0.05). The selective inhibitor of ET-1 subtype B receptor antagonist, BQ788 blocked ET-1 stimulated PDI increases in these cells as was previously reported in red blood cells. Consistent with these results hypoxia was associated with increased mRNA expression of MCP-1, VEGF-a but not ICAM by qRT-PCR and Taqman probes (n=3; P <0.05). Of importance we observed that early cultures of mouse aortic endothelial cells from BERK mice showed similar results. We then evaluated the effects of PDI on erythrocyte hemolysis by exposing cells from patients with SCD to 20 mM 2-2'-azo-bis- (2-amidinopropane) dihydrochloride (AAPH) with or without Q3R or mAb PDI. AAPH-induced hemolysis was dose-dependently blocked by Q3R (IC50: 4.1nM; n=6, P <0.0001 compared to vehicle) or mAb PDI (IC50: 11nM; n=3). Irrelevant IgG did not affect hemolysis under these conditions. Experiments performed in blood from BERK or βSAntilles mice showed similar results. We then studied sickle mice that express varying levels of HbF; BERK (<1% HbF), BERKγM (25% HbF), and BERKγH (45% HbF). BERKγH had the lowest circulating and cell associated PDI activity among the three mouse types that was associated with lower circulating ET-1 levels (n=2; P <0.05). Consistent with these results we observed an inverse correlation between levels of HbF and PDI activity in cells from humans with SCD (n=4). Thus we posit that in SCD elevated erythrocyte PDI activity is important for cell survival and stability and that its inhibition may represent a novel therapeutic approach for improving both the hematological and vascular complications of SCD as it may not only increase sickle erythrocyte survival but may likewise interfere with cellular adhesion leading to reduced vaso-occlusive episodes. [Supported by NIH: HL090632 (AR) and HL096518 (JRR)] Disclosures No relevant conflicts of interest to declare.

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Blockade of the Mineralocorticoid Receptor in Sickle Transgenic Mice Reduces Circulating Protein Disulfide Isomerase and Gardos Channel Activity

Blood ◽

10.1182/blood.v120.21.999.999 ◽

2012 ◽

Vol 120 (21) ◽

pp. 999-999

Author(s):

Christopher Vega ◽

Gregory N. Prado ◽

Philip Asamoah ◽

Patricia Neuman ◽

Jose R Romero ◽

...

Keyword(s):

Endothelial Cells ◽

Transgenic Mice ◽

Protein Disulfide Isomerase ◽

Mineralocorticoid Receptor ◽

Hematological Parameters ◽

Channel Activity ◽

Cell Disease ◽

Disulfide Isomerase ◽

Gardos Channel ◽

Protein Disulfide

Abstract Abstract 999 Endothelin-1 (ET-1), erythrocyte sickling and endothelial cell activation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have provided evidence for the use of ET-1 receptor antagonists in improving hematological parameters in two transgenic mouse models of SCD (Rivera A., 2008, Amer J Physiol). However, the mechanisms that mediate the interplay between red blood cells (RBC) and the endothelium in SCD remain unresolved. Activation of endothelial cells leads to, among other factors, increased levels of protein disulfide isomerase (PDI). PDI catalyzes disulfide interchange reactions, mediates redox modifications and has been observed to be up-regulated under hypoxic conditions. We now report that circulating PDI activity is increased in BERK sickle transgenic mice when compared to wild-type controls. The mineralocorticoid receptor (MR) is a member of the steroid family of nuclear receptors that function as a transcription factor that upon binding to the hormone responsive element of genes such as edn1, the gene for ET-1, leads to increased ET-1 expression. In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and kidney ET-1 mRNA expression. We hypothesized that MR blockade of BERK sickle transgenic mice would lead to reduced PDI activity and improved hematological parameters. Sickle mice were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR receptor antagonist, and tap water ad libitum for 14 days at which time the mice were sacrificed and tissues and blood collected. Plasma PDI activity was calculated by optimization of fluorescently labeled GSSG conversion to GSH. We observed that mice on eplerenone had significantly lower plasma PDI activity than mice on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units; P<0.005, n=6 and 9, respectively). We also studied RBC Gardos channel activity in these mice and observed a significant reduction in clotrimazole-sensitive K+ efflux following MR blockade (2.49±0.5 control and 1.37±0.3 mmol/1013 cells × hr; P<0.04 n= 5 and 7 respectively). MR blockade was associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7) as determined by an ADVIA 120 hematology analyzer. In contrast no significant effects on MCHC levels were observed under these conditions. We then studied ET-1 gene expression using quantitative RT-PCR with ABI Taqman chemistries and GAPDH and β-actin as endogenous controls. We observed that MR blockade was associated with reduced expression of ET-1 mRNA in heart tissue (0.654 ± 0.233, ΔΔCT, relative to mice on regular chow, P<0.04, n=5 and n=7) but not lung tissue. Western blot analyses in membranes from human and mouse sickle erythrocytes and endothelial cells revealed the presence of both MR and PDI proteins. We then studied the effects of ET-1 in early cultures of BERK mouse aortic endothelial cells (MAEC). We observed that stimulation of MAEC cells with 100nM ET-1 for 4 hr was associated with increased mRNA expression of PDI levels that was 1.71 fold greater than vehicle treated cells (n=4, P<0.05). Thus, our results suggest that MR blockade reduces ET-1 levels leading to reduced Gardos and PDI activity in Sickle mice. These effects on PDI activity and Gardos channel regulation may represent a novel mechanism for protective effects of MR blockade aimed at ameliorating vascular complications of Sickle Cell Disease. Supported by NIH R01HL090632 to AR. Disclosures: No relevant conflicts of interest to declare.

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Aldosterone Stimulates Neutrophils Leading To Increased β-Glucuronidase, Protein Disulfide Isomerase and Myeloperoxidase Secretion

Blood ◽

10.1182/blood.v122.21.2271.2271 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2271-2271

Author(s):

Arelys Ramos-Rivera ◽

Alicia Rivera ◽

Enrique D. Machado-Fiallo ◽

Josue A. Benabe-Carlo ◽

Gregory N. Prado ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Respiratory Burst ◽

Protein Disulfide Isomerase ◽

Mrna Levels ◽

Dependent Manner ◽

Cell Disease ◽

Rt Pcr ◽

Disulfide Isomerase ◽

Protein Disulfide

Abstract Aldosterone (ALDO) has been shown to play an important role in inflammatory responses in addition to its well described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). However, its effects on polymorphonuclear leukocytes (PMNC) are not well described. We isolated untouched circulating human PMNC by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% of PMNC were positive for the myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses. We incubated PMNC with ALDO (10–9–10–7M) for 30 min and observed a dose-dependent rise in β–glucuronidase release with an EC50 of 6.11 nM (P<0.001, n=3), an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). In addition, our results show that incubation of human PMNC with 10-8M ALDO likewise led to increases in myeloperoxidase ([MPO], P<0.05, n=3) and protein disulfide isomerase ([PDI], P<0.01, n=4), a multifunctional enzyme of the thioredoxin superfamily that mediates redox modifications, regulates KCNN4 channel and erythrocyte volume and is up-regulated under hypoxic conditions (Prado, 2013 FASEB J). We then studied the effects of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 5 days with 1.3% DMSO. Our results likewise show an increase in MPO responses upon 10–8M ALDO stimulation as compared to vehicle (AUC: 1090±147 to 505±48, P<0.02, n=3). We have recently reported that aldosterone stimulates increases of striatin, a scaffolding protein that interacts with caveolin-1, and co-precipitates with striatin and as such may facilitate cross talk of signaling complexes. As there are no pharmacological inhibitors of striatin we used a molecular approach to reduce striatin levels. In differentiated HL-60 cells, siRNA against striatin led to reduced MPO responses (AUC: 590±14 to 528±13, P<0.05, n=3) that were associated with significantly reduced striatin mRNA levels but not when cells were transfected with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and β-microglobulin used as an endogenous control (P<0.01, n=3). These results suggest that striatin plays an important role in ALDO-stimulated degranulation responses. Of importance we also observed that incubation with ALDO (10–9–10–7M) in differentiated HL60 cells led to increases in the oxidative-respiratory burst [superoxide production] in a dose- and time-dependent manner (P<0.01, n=4). Consistent with these results, we observed that ALDO likewise led to significant increases in the oxidative-respiratory burst in human PMNC (P<0.01, n=3). As there is evidence that activated neutrophils, MPO and PDI are elevated in Sickle Cell Disease, we studied the in vivo effects of MR blockade in BERK sickle transgenic mice, a model of increased oxidative stress. Sickle mice were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR receptor antagonist, and tap water ad libitum for 14 days at which time the mice were sacrificed and blood collected. We observed that mice on eplerenone had significantly lower plasma PDI activity than mice on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units [RFU]; P<0.005, n=6 and 9) and lower MPO levels (AUC: 214±11 to 73±20, P<0.03, n=3); events that were associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). Thus, our results suggest that MR activation by ALDO is a novel mechanism for neutrophil stimulation and as such represents a novel therapeutic target aimed at ameliorating the vascular complications of Sickle Cell Disease. Supported by NIH R01HL090632 (AR) and R01HL096518 (JRR). Disclosures: No relevant conflicts of interest to declare.

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Regulation of Erythrocyte Volume by Cell Surface Associated Protein Disulfide Isomerase.

Blood ◽

10.1182/blood.v110.11.149.149 ◽

2007 ◽

Vol 110 (11) ◽

pp. 149-149

Author(s):

Jose R. Romero ◽

Manuel D. Bicho ◽

Lin-Chien Pong ◽

Joel Greenbowe ◽

Alicia Rivera

Keyword(s):

Sickle Cell Disease ◽

Cell Surface ◽

Sickle Cell ◽

Protein Disulfide Isomerase ◽

Red Cells ◽

Channel Activity ◽

Cell Disease ◽

Gardos Channel ◽

Sickle Erythrocytes ◽

Protein Disulfide

Abstract Acute and chronic clinical manifestations of sickle cell disease (SCD) are based on vaso-occlusion and impaired blood flow. Dense erythrocytes are believed to be important contributors to the vaso-occlusive manifestations of SCD. However, the physiological regulation of erythrocyte hydration status in SCD is not entirely clear. The Gardos channel and K/Cl cotransport are major contributors to sickle erythrocyte dehydration. Protein disulfide isomerase (PDI) on the cell surface catalyzes disulfide formation, causes redox modifications and has been observed to be up-regulated under hypoxic conditions. We now report the detection of PDI in red cells. Western blot analysis revealed a prominent band, migrating at 55 kDa, in ghost preparation from both sickle and normal erythrocytes. To evaluate the role of PDI in Gardos channel activation, we measured charybdotoxin-sensitive K+ influx in the presence of bacitracin, a well-known blocker of PDI activity. When sickle erythrocytes are exposed to bacitracin, Gardos channel activity is significantly reduced (1.4 ±0.2 to 0.8± 0.05 mmol/L cell x min, n=6, P<0.01). We also observed that Gardos channel activity was attenuated by 0.25 mM bacitracin and was maximally inhibited by 3 mM in sickle and normal erythrocytes. Similar results were observed with phenyl arsine oxide and acetyl-thyroxin, two other well-known inhibitors of cell surface PDI activity. We then studied the effects of PDI inhibition on red cell density profiles of sickle and normal human red cells. Analysis of the baseline density profile indicates that erythrocytes have a median density of 1.10 g/mL. This value significantly increased to 1.125 g/mL (n=2) after 3 h of oxygenation/de-oxygenation cycles. However, in the presence of 3 mM bacitracin, the cellular density profile markedly shifted to the left (1.098 g/mL) following deoxygenation/oxygenation. We also investigated the reductive capacity of freshly isolated human erythrocytes by the ferrocyanide-production method. The reductive capacity of normal Hb A red cells was significantly lower than in Hb S containing cells (2.8 ± 1.1 vs 5.1 ± 1.3 mmol/L cell x h, n=18, p<0.0001). Similar results were observed in Santillies and NYKO1, two transgenic mouse models of sickle cell disease, when compared to C57 mice. These results strongly implicate cell surface associated PDI in cellular dehydration and formation of dense sickle erythrocytes by activating K+ efflux via the Gardos channel and suggest that aberrant regulation of PDI activity and/or its expression may contribute to the pathophysiology of Sickle Cell Disease.

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Endothelium but Not Platelet-Derived Protein Disulfide Isomerase Is Required for Fibrin Generation during Thrombus Formation in Vivo.

Blood ◽

10.1182/blood.v112.11.691.691 ◽

2008 ◽

Vol 112 (11) ◽

pp. 691-691 ◽

Cited By ~ 1

Author(s):

Reema Jasuja ◽

Jaehyung Cho ◽

Bruce Furie ◽

Barbara Furie

Keyword(s):

Endothelial Cells ◽

Protein Disulfide Isomerase ◽

Thrombus Formation ◽

Fibrin Deposition ◽

Wild Type ◽

Disulfide Isomerase ◽

Laser Injury ◽

Injury Model ◽

Protein Disulfide

Abstract We have previously reported that protein disulfide isomerase is required in wild-type mice for platelet thrombus formation and fibrin generation in an in vivo laser injury model of thrombosis (Cho et al. J. Clin. Invest., 2008; 118:1123–31). Fibrin deposition after laser injury to the vessel wall in Par4−/− mice, lacking the G protein-coupled platelet thrombin receptor, is independent of platelets or requires minimal platelet activation or accumulation (Vandendries et al. Proc. Natl. Acad. Sci., 2007; 104:288–92). However, protein disulfide isomerase inhibitors have a dramatic effect on fibrin accumulation in Par4− mice, suggesting that these inhibitors may function by a platelet independent mechanism. Here, we compare the contributions of endothelium and platelet-derived protein disulfide isomerase to fibrin generation in the mouse laser injury model of thrombosis. In vitro studies using cultured human umbilical vein endothelial cells and human aortic endothelial cells show that protein disulfide isomerase can be secreted rapidly into the culture medium from these cells upon thrombin stimulation. Using intravital microscopy, we observe that protein disulfide isomerase is not detectable on the vessel wall prior to laser injury but can be detected on the injured cremaster arteriolar wall and in the developing thrombus very rapidly after laser induced injury in the live mouse. The median integrated fluorescence intensity for protein disulfide isomerase in wild-type mice was compared to wild-type mice injected with 10ug/g mouse of Integrilin, an inhibitor of platelet activation and platelet thrombus formation, and thus, an inhibitor of the contribution of platelet derived protein disulfide isomerase to thrombus formation. Protein disulfide isomerase expression was similar in both treated and untreated animals upto 30 seconds post-laser injury. After 30 seconds, the expression of protein disulfide isomerase in integrilin treated mice was significantly decreased compared to that in untreated mice, indicating that the initial protein disulfide isomerase was derived from the endothelium and later additional protein disulfide isomerase was derived from the platelets following their accumulation in the developing thrombus. Fibrin deposition, a measure of thrombin generation was comparable in wild-type mice that had been treated with Integrilin or treated with a control buffer, suggesting that endothelial-derived protein disulfide isomerase was sufficient for fibrin generation. The rate and amount of fibrin generation was indistinguishable in both groups. Furthermore, inhibition of the protein disulfide isomerase with the function blocking monoclonal antibody RL-90 (3ug/g mouse) eliminated any fibrin deposition in wild-type mice that had been treated with Integrilin. Taken together, these data indicate that endothelium-derived protein disulfide isomerase is necessary to support fibrin deposition in vivo in our laser injury model of thrombus formation. The initial protein disulfide isomerase expressed at the site of injury is derived from endothelial cells but platelets activated at the site of thrombus formation contribute, amplify and sustain protein disulfide isomerase expression.

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Endothelium-derived but not platelet-derived protein disulfide isomerase is required for thrombus formation in vivo

Blood ◽

10.1182/blood-2010-04-278184 ◽

2010 ◽

Vol 116 (22) ◽

pp. 4665-4674 ◽

Cited By ~ 94

Author(s):

Reema Jasuja ◽

Bruce Furie ◽

Barbara C. Furie

Keyword(s):

Endothelial Cells ◽

Protein Disulfide Isomerase ◽

Vessel Wall ◽

Thrombus Formation ◽

In Vivo Studies ◽

Disulfide Isomerase ◽

Platelet Thrombus ◽

Wall Injury ◽

Protein Disulfide

Protein disulfide isomerase (PDI) catalyzes the oxidation reduction and isomerization of disulfide bonds. We have previously identified an important role for extracellular PDI during thrombus formation in vivo. Here, we show that endothelial cells are a critical cellular source of secreted PDI, important for fibrin generation and platelet accumulation in vivo. Functional PDI is rapidly secreted from human umbilical vein endothelial cells in culture upon activation with thrombin or after laser-induced stimulation. PDI is localized in different cellular compartments in activated and quiescent endothelial cells, and is redistributed to the plasma membrane after cell activation. In vivo studies using intravital microscopy show that PDI appears rapidly after laser-induced vessel wall injury, before the appearance of the platelet thrombus. If platelet thrombus formation is inhibited by the infusion of eptifibatide into the circulation, PDI is detected after vessel wall injury, and fibrin deposition is normal. Treatment of mice with a function blocking anti-PDI antibody completely inhibits fibrin generation in eptifibatide-treated mice. These results indicate that, although both platelets and endothelial cells secrete PDI after laser-induced injury, PDI from endothelial cells is required for fibrin generation in vivo.

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

Blood ◽

10.1182/blood.v122.21.12.12 ◽

2013 ◽

Vol 122 (21) ◽

pp. 12-12

Author(s):

Josue A. Benabe-Carlo ◽

Jose R Romero ◽

Enrique D. Machado-Fiallo ◽

Arelys Ramos-Rivera ◽

Gregory N. Prado ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Disulfide Isomerase ◽

Conflicts Of Interest ◽

Mean Cell Volume ◽

Distribution Width ◽

Disulfide Isomerase ◽

Erythrocyte Volume ◽

Caveolin 1 ◽

Protein Disulfide

Abstract 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). Disclosures: No relevant conflicts of interest to declare.

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Treatment with NS3623, a novel Cl-conductance blocker, ameliorates erythrocyte dehydration in transgenic SAD mice: a possible new therapeutic approach for sickle cell disease

Blood ◽

10.1182/blood.v97.5.1451 ◽

2001 ◽

Vol 97 (5) ◽

pp. 1451-1457 ◽

Cited By ~ 48

Author(s):

Poul Bennekou ◽

Lucia de Franceschi ◽

Ove Pedersen ◽

Lurong Lian ◽

Toshio Asakura ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Hemoglobin Concentration ◽

Repeated Administration ◽

K Channel ◽

Mean Corpuscular Hemoglobin ◽

Cell Disease ◽

Cl Conductance

The dehydration of sickle red blood cells (RBCs) through the Ca-activated K channel depends on the parallel movement of Cl ions. To study whether Cl-conductance block might prevent dehydration of sickle RBCs, a novel Cl-conductance inhibitor (NS3623) was characterized in vitro using RBCs from healthy donors and sickle cell patients and in vivo using normal mice and a transgenic mouse model of sickle cell disease (SAD mice). In vitro, NS3623 reversibly blocked human RBC Cl-conductance (gCl) with an IC50 value of 210 nmol/L and a maximal block of 95%. In vivo, NS3623 inhibited RBC gCl after oral administration to normal mice (ED50 = 25 mg/kg). Although gCl, at a single dose of 100 mg/kg, was still 70% inhibited 5 hours after dosing, the inhibition disappeared after 24 hours. Repeated administration of 100 mg/kg twice a day for 10 days caused no adverse effects; therefore, this regimen was chosen as the highest dosing for the SAD mice. SAD mice were treated for 3 weeks with 2 daily administrations of 10, 35, and 100 mg/kg NS3623, respectively. The hematocrit increased, and the mean corpuscular hemoglobin concentration decreased in all groups with a concomitant increase in the intracellular cation content. A loss of the densest red cell population was observed in conjunction with a shift from a high proportion of sickled to well-hydrated discoid erythrocytes, with some echinocytes present at the highest dosage. These data indicate feasibility for the potential use of Cl-conductance blockers to treat human sickle cell disease.

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