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

Blood ◽  
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.

Protein Disulfide Isomerase in Sickle Cell Disease

Blood ◽  
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.

scholarly journals Activation of the Mineralocorticoid Receptor Stimulates Protein Disulfide Isomerase: Role in Sickle Cell Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1351-1351
Author(s):  
Alexandra Lozano ◽  
Christopher Vega ◽  
Yaritza Inostroza-Nieves ◽  
Lorena Rivera González ◽  
Pablo J. López ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Protein Disulfide Isomerase ◽  
Mineralocorticoid Receptor ◽  
Conflicts Of Interest ◽  
Hematological Parameters ◽  
Control Diet ◽  
Mrna Levels ◽  
Disulfide Isomerase ◽  
Qrt Pcr ◽  
Protein Disulfide

Abstract Activation of the mineralocorticoid receptor (MR), a critical component of the Renin-Angiotensin-Aldosterone (ALDO)-System (RAAS), has been shown to play an important role in inflammatory and vascular endothelial responses in addition to its well-described effects on sodium and water homeostasis. Activationof endothelial cells leads to, among other factors, increased endothelin-1 (ET-1) and protein disulfide isomerase (PDI) release. PDI and ET-1 contribute to vascular inflammation and are increased in patients with SCD and sickle transgenic mouse models. The MR is a member of the steroid family of nuclear receptors and transcription factors that upon activation binds to hormone response elements of edn1, the gene for ET-1, leading to increased ET-1 expression.In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and ET-1 mRNA expression. However, the role of MR in SCD is unclear. We hypothesized that MR blockade in sickle transgenic mice would reduce PDI activity and improve hematological parameters and inflammation. We first studied EA.hy926 (EA) cells, a human endothelial cell line that expresses MR. We incubated EA cells with ALDO (10-8 M), an MR agonist, for 24 hr and observed a rise in PDI mRNA levels by qRT-PCR (P<0.01, n=5), an event that was blocked by pre-incubation of EA cells with 1 μM canrenoic acid (CA), an MR antagonist (P<0.05, n=5). We then measured PDI activity in the supernatant of ALDO-stimulated EA cells using a Di-E-GSSH fluorescent marker and observed a rise in PDI activity following ALDO (10-8 M) when compared to vehicle treatment (P<0.05; n=5). To test the in vivo effects of MR activation, we studied Berkeley Sickle Transgenic (BERK) mice that were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR antagonist (MRA), for 14 days. We observed significantly lower plasma PDI activity in mice treated with MRA than those 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, respectively). Treatment with MRA was associated with reduced plasma ET-1 and myeloperoxidase (MPO) levels in BERK mice. 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 x hr; P<0.04 n= 5 and 7 respectively). Consistent with these results, 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). We also studied gene expression by qRT-PCR in heart tissue from these mice and observed that MR blockade reduced mRNA expression of: ET-1 (0.654 ± 0.233, P<0.05, n=5 and n=7); PDI (0.546 ± 0.063, P<0.01, n=5 and n=7); and Tumor Necrosis Factor Receptor Superfamily Member 1A mRNA (0.464 ± 0.061, P<0.01, n=5 and n=7). Thus, our results suggest a novel role for RAAS and, in particular, MR activation in SCD. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1080-1080
Author(s):  
Giselle Brito ◽  
Gregory N. Prado ◽  
Sonia E. Henriquez ◽  
Iren M. Ortiz ◽  
Carlos E Vazquez ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell ◽  
Protein Disulfide Isomerase ◽  
K Channel ◽  
Channel Activity ◽  
Cell Disease ◽  
Rt Pcr ◽  
Endogenous Controls ◽  
Protein Disulfide

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.

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

Blood ◽  
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&lt;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&lt;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.

Abstract 776: Protein Disulfide Isomerase Is Involved In Redox Regulation Of Nitric Oxide Output During Laminar Shear Stress In Endothelial Cells

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Denise C Fernandes ◽  
Celio X Santos ◽  
Hanjoong Jo ◽  
Francisco R Laurindo
Keyword(s):  
Endothelial Cells ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Membrane Fraction ◽  
Protein Disulfide Isomerase ◽  
Disulfide Isomerase ◽  
Nadph Oxidase Activity ◽  
Caveolin 1 ◽  
Protein Disulfide ◽  
Laminar Shear

While anti-atherogenic effects of sustained laminar shear (LS) involve NO release from eNOS, increases in LS trigger transient superoxide production via NADPH oxidase. Recently, we showed that NADPH oxidase undergoes thiol-dependent regulation by the thioredoxin superfamily chaperone Protein Disulfide Isomerase (PDI). PDI is known to promote NO internalization via trans-nitrosation reactions. We hypothesized that PDI-dependent support of NADPH oxidase activity affects NO output during sustained LS. Cultured rabbit aortic endothelial cells (RAEC) submitted to LS (15 dynes/cm 2 ) in a cone-plate system for 18h exhibited (vs. static controls): Decreased (~50%) superoxide production (HPLC analysis of DHE oxidation); Decreased (~20%) NADPH-triggered hydrogen peroxide production in membrane fraction (Amplex Red assay); Decreased mRNA expression of Nox1 (67%) and Nox4 (45%) (real-time QPCR); Increased eNOS expression (~50%, western blot) and nitrite levels in culture medium (Δ = 7.1±2.5[SD] μM, NO Analyzer and Griess reaction); Decrease in total and membrane fraction PDI protein expression (~20%) without changes in membrane fraction/total ratio of PDI. RAEC were transfected with c-myc -tagged plasmid coding for wild-type (WT) PDI or PDI mutated in 4 thioredoxin-motif cysteine residues. Forced expression (2-fold) of mutated but not WT PDI led to increase in nitrite output after LS (18h) (Δmutated = 17.2±3.3 μM vs. ΔWT = 7.0±1.9 μM, n=3, p<0.02). Confocal microscopy indicated similar subcellular localization between WT and mutated PDI. PDI co-imunoprecipitated with p22phox NADPH oxidase subunit, but not with eNOS or caveolin-1, either in static condition or after LS. Fractionation studies in sucrose gradients showed that PDI is distributed throughout several fractions in static conditions, including caveolin-1-enriched fractions, but migrates to higher-density fractions, not containing caveolin-1, during sustained LS. These results suggest that PDI is involved in regulation of NO output during LS via its effects on NADPH oxidase activity.

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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 691-691 ◽  
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.

Aldosterone Stimulates a Degranulation Response in Human Neutrophils: Role of Protein Disulfide Isomerase

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1034-1034
Author(s):  
Daphne Diaz ◽  
Gregory N. Prado ◽  
Patricia Neuman ◽  
Adriana Nieva ◽  
Manuel Torres-Grajales ◽  
...  
Keyword(s):  
Western Blot ◽  
Protein Disulfide Isomerase ◽  
Mineralocorticoid Receptor ◽  
Inflammatory Responses ◽  
Rt Pcr ◽  
Disulfide Isomerase ◽  
Net Production ◽  
Protein Disulfide ◽  
Dose Dependent

Abstract Abstract 1034 There is growing evidence for an important role of aldosterone (ALDO) in inflammatory responses in addition to its well-described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). We studied the effects of ALDO on activation of ex vivo human polymorphonuclear leukocytes (PMNC). 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 myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses and when incubated with ALDO (10−9 −10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 2 min using FURA-2AM fluorescence. We then studied the effect of ALDO on PMNC degranulation following incubations with ALDO (10−9 −10−7 M) for 30 min and observed a significant increase in β–glucuronidase release (P<0.001, n=3) by established fluorescent detection methods, an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). PMA and N-Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) were used as positive controls for PMNC activation. 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. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcripts by quantitative RT-PCR using TaqMan detection probes in these cells and as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence. To assess the degranulation response of these cells we quantified the in vitro release of myeloperoxidase (MPO) and observed that 10−8M ALDO was likewise associated with increased degranulation when compared to vehicle treated cells (AUC: 590±14 to 185±11, P<0.01, n=6). To characterize the mechanisms by which ALDO regulates the degranulation responses of these cells we studied the effects of Protein Disulfide Isomerase (PDI) on ALDO-stimulated cells. PDI catalyzes the oxidation or reduction of thiol/disulfide groups and modulates leukocyte function. Our results show that blockade of PDI, by bacitracin, led to a blunted ALDO-stimulated degranulation response in both cell types. Consistent with these observations, we show that in differentiated HL-60 cells, siRNA against PDI likewise led to reduced MPO responses (AUC: 590±14 to 290±13, P<0.01, n=6) that were associated with significantly reduced PDI mRNA levels but not with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and GAPDH and β2 microglobulin as endogenous controls (0.55 ± 0.02, ΔΔCT of PDI siRNA relative to scrambled transfected cells, P<0.01, n=6). These results suggest that ALDO stimulates MPO release. MPO has been shown to be one of the predominant granule proteins associated with Neutrophil Extracelullar Traps (NETs), extracellular structures that contain chromatin (DNA and histones) that can also trap microorganisms. We studied the effects of ALDO following digestion of the NETs by DNAse, and observed that 30–35% of the total cellular MPO was NET-associated. We also observed that incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3), a responses that was blocked by pre-incubation of cells with 1 uM CA (P<0.03, n=3). 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). Thus our results suggest that activation of MR by ALDO leads to degranulation and NET production in neutrophils that may contribute to the inflammatory responses associated with MR activation in vivo. Furthermore, the association between degranulation and NET release implicates PDI as a novel regulator of MPO generated NET production. Disclosures: No relevant conflicts of interest to declare.

Extracellular Protein Disulfide Isomerase Regulates Vitronectin during the Initiation of Thrombus Formation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 15-15
Author(s):  
Chao Fang ◽  
Sheryl R. Bowley ◽  
Barbara C. Furie ◽  
Bruce Furie
Keyword(s):  
Endothelial Cells ◽  
Protein Disulfide Isomerase ◽  
Vessel Wall ◽  
Plasma Sample ◽  
Thrombus Formation ◽  
Disulfide Isomerase ◽  
Platelet Accumulation ◽  
Protein Disulfide ◽  
Kinetic Trapping

Abstract Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells upon vascular injury, is required for thrombus formation. However, the precise mechanism by which PDI regulates thrombosis remains elusive. Using PDI variants that form stable mixed disulfide complexes with their substrates, we performed kinetic trapping experiment in platelet rich plasma and identified multiple substrate proteins for PDI, including vitronectin. Importantly, when using variants of endoplasmic reticulum protein 57 (ERp57), a thiol isomerase that has a similar domain structure as PDI and is also important for thrombus formation, the trapping mutants of ERp57 do not interact with vitronectin. This result has demonstrated the substrate specificity of PDI during our kinetic trapping experiment. Further study using polyethylene glycol (PEG)-based gel mobility shift assay combined with mass spectrometry has identified the redox reaction between PDI and vitronectin occurs on two disulfide bonds Cys 137-161 and Cys 274-453 in the hemopexin-like domains of plasma vitronectin. Vitronectin, as a substrate of extracellular PDI, has been shown to be important for thrombus formation. Vitronectin null mice have reduced platelet accumulation and fibrin deposition in the cremaster arterioles following laser injury. Vitronectin null mice also have significantly prolonged large-vessel thrombosis in the carotid artery using the ferric chloride thrombosis model. Using intravital microscopy we showed that vitronectin rapidly accumulates in a growing thrombus following vessel injury. When mice are treated with eptifibatide to eliminate platelet accumulation, we still observe significant amount of vitronectin accumulation on the vessel wall in the absence of platelet thrombus. This observation was further confirmed using confocal intravital microscopy. After 3D reconstruction of a growing thrombus in mouse cremaster arteriole, vitronectin was identified to locate primarily on the CD31 stained vessel wall. These combined studies suggest that plasma-derived vitronectin and not platelet-derived vitronectin is the primary substrate of PDI. Our study further showed that the indispensable role of vitronectin to a growing thrombus depends on extracellular PDI. Native plasma vitronectin does not bind to αvβ3 or αIIbβ3-integrins on endothelial cells and platelets. On solid phase binding assay, plasma sample pre-treated with wild-type PDI showed significantly increased binding of vitronectin to its ligand αvβ3 or αIIbβ3-integrins. However, this increase was not observed in plasma pre-treated with dead-mutant PDI or ERp57. In addition, using immunofluorescent staining, PDI treated plasma sample also showed significantly increased binding of vitronectin to activated human umbilical vein endothelial cells (HUVECs) and this binding was abrogated by RGD peptides or an αvβ3 blocking antibody. The critical role of extracellular PDI for the regulation of vitronectin in a growing thrombus was further confirmed in our in vivo studies. When mice were treated with quecetin-3-rutinoside or two different inhibitory antibodies that selectively block PDI activity, the accumulation of vitronectin and platelets was significantly reduced. These combined results demonstrate that extracellular PDI regulates vitronectin in a growing thrombus to promote platelet accumulation and fibrin generation. In summary, our studies have revealed a novel regulatory mechanism during the initiation of thrombus formation. Under normal physiologic conditions in the absence of secreted PDI, thrombus formation is suppressed and maintains a quiescent, patent vasculature. The release of PDI during vascular injury serves as a novel regulatory switch that allows activation of proteins, including vitronectin, which are critical for the following platelet accumulation and fibrin generation. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2010 ◽  
Vol 116 (22) ◽  
pp. 4665-4674 ◽  
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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