Protein Disulfide Isomerase Expression Research Articles

Inhibition of Protein Disulfide Isomerase Attenuates Osteoclast Differentiation and Function via the Readjustment of Cellular Redox State in Postmenopausal Osteoporosis.

Due to the accumulation of reactive oxygen species (ROS) and heightened activity of osteoclasts, postmenopausal osteoporosis could cause severe pathological bone destruction. Protein disulfide isomerase (PDI), an endoplasmic prototypic thiol isomerase, plays a central role in affecting cellular redox state. To test whether suppression of PDI could inhibit osteoclastogenesis through cellular redox regulation, bioinformatics network analysis was performed on the causative genes, followed by biological validation on the osteoclastogenesis in vitro and ovariectomy (OVX) mice model in vivo. The analysis identified PDI as one of gene targets for postmenopausal osteoporosis, which was positively expressed during osteoclastogenesis. Therefore, PDI expression inhibitor and chaperone activity inhibitor were used to verify the effects of PDI inhibitors on osteoclastogenesis. Results demonstrated that PDI inhibitors could reduce osteoclast number and inhibit resorption function via suppression on osteoclast marker genes. The mechanisms behind the scenes were the PDI inhibitors-caused intracellular ROS reduction via enhancement of the antioxidant system. Micro-CT and histological results indicated PDI inhibitors could effectively alleviate or even prevent bone loss in OVX mice. In conclusion, our findings unveiled the suppressive effects of PDI inhibitors on osteoclastogenesis by reducing intracellular ROS, providing new therapeutic options for postmenopausal osteoporosis.

CDDO-Me Abrogates Aberrant Mitochondrial Elongation in Clasmatodendritic Degeneration by Regulating NF-κB-PDI-Mediated S-Nitrosylation of DRP1

Clasmatodendrosis is a kind of astroglial degeneration pattern which facilitates excessive autophagy. Although abnormal mitochondrial elongation is relevant to this astroglial degeneration, the underlying mechanisms of aberrant mitochondrial dynamics are still incompletely understood. Protein disulfide isomerase (PDI) is an oxidoreductase in the endoplasmic reticulum (ER). Since PDI expression is downregulated in clasmatodendritic astrocytes, PDI may be involved in aberrant mitochondrial elongation in clasmatodendritic astrocytes. In the present study, 26% of CA1 astrocytes showed clasmatodendritic degeneration in chronic epilepsy rats. 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me; bardoxolone methyl or RTA 402) and SN50 (a nuclear factor-κB (NF-κB) inhibitor) ameliorated the fraction of clasmatodendritic astrocytes to 6.8 and 8.1% in CA1 astrocytes, accompanied by the decreases in lysosomal-associated membrane protein 1 (LAMP1) expression and microtubule-associated protein 1A/1B light-chain 3 (LC3)-II/LC3-I ratio, indicating the reduced autophagy flux. Furthermore, CDDO-Me and SN50 reduced NF-κB S529 fluorescent intensity to 0.6- and 0.57-fold of vehicle-treated animal level, respectively. CDDO-Me and SN50 facilitated mitochondrial fission in CA1 astrocytes, independent of dynamin-related protein 1 (DRP1) S616 phosphorylation. In chronic epilepsy rats, total PDI protein, S-nitrosylated PDI (SNO-PDI), and SNO-DRP1 levels were 0.35-, 0.34- and 0.45-fold of control level, respectively, in the CA1 region and increased CDDO-Me and SN50. Furthermore, PDI knockdown resulted in mitochondrial elongation in intact CA1 astrocytes under physiological condition, while it did not evoke clasmatodendrosis. Therefore, our findings suggest that NF-κB-mediated PDI inhibition may play an important role in clasmatodendrosis via aberrant mitochondrial elongation.

Abstract 517: Protein disulfide isomerase as a predictive biomarker for non-muscle invasive bladder cancer recurrence

Abstract INTRODUCTION AND OBJECTIVE: High recurrence rates found in non-muscle invasive bladder cancer (NMIBC) pose a financial and psychological burden for patients. We demonstrated that bladder cancer (BC) cells release protein disulfide isomerase (PDI), a redox-regulated ER-resident protein, via extracellular vesicles (EVs) when experiencing high levels of ER stress for their own survival. Chronic exposure to PDI-enriched BC EVs induced malignant transformation in recipient non-transformed cells. We hypothesized that the dynamic redox state in BC cells determines PDI cellular localization and tumors with elevated PDI preferentially incorporate PDI into EVs for removal. Furthermore, PDI expression levels and subcellular localization in tumors from NMIBC patients may predict NMIBC recurrence. METHODS: Retrospective chart review identified 122 patients with newly diagnosed low-grade NMIBC classified into 2 groups: recurrent (N=55) and non-recurrent (N=67) during follow-up period. Formalin fixed paraffin embedded tumor specimens from index transurethral resection of bladder tumor (TURBT) were obtained and a tissue microarray (TMA) was constructed. Immunofluorescent staining of TMA sections was conducted using antibodies against PDI and BiP, an ER stress marker. Stained tissues were photographed and tumor regions in each field were manually masked and confirmed by a genitourinary pathologist. Within each tumor region, total antibody labeling was determined by measuring the mean pixel intensity values with NIH ImageJ/Fiji. Manders coefficient was used to assess the colocalization of epitopes using JACoP plugin in ImageJ. RESULTS: Tumor tissues from patients that recurred expressed significantly higher levels of PDI and BiP. Moreover, high PDI and BiP expression predicted worse recurrence-free survival. These data suggest that tissues under higher levels of ER stress—as indicated by higher levels of PDI and BiP expression—have a higher risk of recurrence. Significantly higher levels of PDI were detected in non-ER cytosol in recurrent cohort tumors. CONCLUSIONS: This study provides a novel mechanism for BC recurrence and valuable clinical predictive biomarkers for NMIBC recurrence and progression. Future clinical applications targeting this pathway may be an avenue to prevent NMIBC recurrence. Citation Format: Kit L. Yuen, Chia-Hao Wu, Christopher R. Silvers, Alexis R. Steinmetz, Hiroshi Miyamoto, Edward M. Messing, Yi-Fen Lee. Protein disulfide isomerase as a predictive biomarker for non-muscle invasive bladder cancer recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 517.

Sigma‑1 receptor overexpression promotes proliferation and ameliorates cell apoptosis in β‑cells.

Sigma-1 receptor (Sig-1R) is a class of orphan receptors, the potential role of which in pancreatic islet cells remains poorly understood. The present study aimed to investigate the role of Sig-1R in islet β-cell proliferation and examine the effects of Sig-1R on islet β-cell injury under lipotoxic conditions. Sig-1R-overexpressing MIN6 cells were generated by lentiviral vector transfection. The effect of Sig-1R overexpression on cell proliferation detected by EdU staining, cell cycle progression by propidium iodide (PI), apoptosis by Annexin V-APC/PI, mitochondrial membrane potential by Mitolite Red and cytoplasmic Ca2+ levelsby Fura-2/AM in islet β-cells were measured by flow cytometry. Western blot analysis was used to measure protein expression levels of endoplasmic reticulum (ER) stress markers glucose-regulated protein 78 and C/EBP homologous protein, mitochondrial apoptotic proteins Bcl-2-associated X and Bcl-2 and cytochrome c. In addition, ATP levels and insulin secretion were separately measured using ATP Assay and mouse insulin ELISA. Mitochondria-associated ER membrane (MAM) structures in MIN6 cells were then detected using transmission electron microscopy. Protein disulfide isomerase expression and possible colocalization between inositol 1,4,5-trisphosphate receptor and voltage-dependent anion channel 1 were examined using immunofluorescence. Sig-1R overexpression was found to promote β-cell proliferation by accelerating cell cycle progression. Furthermore, Sig-1R overexpression ameliorated the apoptosis rate whilst impairing insulin secretion induced by palmitic acid by relieving ER stress and mitochondrial dysfunction in MIN6 cells. Sig-1R overexpression also promoted Ca2+ transport between mitochondria and ER by increasing the quantity of ER adjacent to mitochondria in the 50-nm range. It was concluded that Sig-1R overexpression conferred protective effects on β-cells against lipotoxicity as a result of the promotion of cell proliferation and inhibition of ER stress and oxidative stress, by regulating the structure of MAM.

Effects of protein disulfide isomerase on hyperglycemia and hypoxia/reoxygenation injury in H9c2 cardiomyocytes

Objective: To explore the effect of protein disulfide isomerase (PDI) in diabetic ischemic heart disease. Methods: We established an in vitro model of high glucose and hypoxia/reoxygenation in H9c2 rat myocardial cells. Cultured cells were divided into four groups: Control, high glucose (HG), hypoxia/reoxygenation (H/R) and HG+H/R. Changes in PDI expression mediated by PDI adenovirus(Ad-PDI) infection and siRNA(PDI-siRNA) transfection in myocardial cells were observed by inverted fluorescence microscopy. We also measured lactate dehydrogenase(LDH) activity and malondialdehyde(MDA) and high molecular weight(HMW)-APN concentrations. PDI, APN, cleaved caspase-3, and glucose regulated protein 78 (Grp78) protein expression were detected. Results: PDI expression was significantly decreased in the HG, H/R and HG+H/R groups compared to the Control group; however, LDH activity[(179.7±10.4) U/L、(218.4±18.4) U/L、(328.2±5.3) U/L vs (91.0±11.0) U/L], MDA concentration[(7.0±0.4) μmol/L、(10.0±1.0) μmol/L、(11.7±1.0) μmol/L vs (4.2±1.8) μmol/L], cleaved caspase-3, and Grp78 expression were increased. Interestingly, APN and HMW-APN expression were decreased [(2.01±0.21) μg/L、(1.64±0.27) μg/L、(1.20±0.14) μg/L vs (2.62±0.12) μg/L, all P<0.05]. Over expression of PDI attenuated high glucose and hypoxia/reoxygenation induced apoptosis and oxidative stress in H9c2 cardiomyocytes(all P<0.05), and simultaneously increased APN and HMW-APN expression [(2.86±0.03) μg/L vs (3.03±0.10) μg/L、(2.06±0.05) μg/L vs (2.31±0.06) μg/L、(1.83±0.07) μg/L vs (1.96±0.11) μg/L、(1.20±0.06) μg/L vs (1.39±0.09) μg/L]. PDI-siRNA transfection increased LDH activity, MDA concentration, and cleaved caspase-3 and Grp78 expression, and decreased APN and HMW-APN expression [(0.75±0.09) μg/L vs (0.59±0.09) μg/L、(0.62±0.04) μg/L vs (0.53±0.05) μg/L、(0.55±0.14) μg/L vs (0.51±0.12) μg/L、(0.48±0.12) μg/L vs (0.35±0.08) μg/L] in response to different treatments in cultured H9c2 cardiomyocytes (all P<0.05). Conclusion: PDI may regulate the expression of APN and HMW-APN, and play an important role in the function of diabetic ischemia-reperfusion cardiomyocytes.

Protein Disulfide Isomerases Regulate IgE-Mediated Mast Cell Responses and Their Inhibition Confers Protective Effects During Food Allergy.

The thiol isomerase, protein disulfide isomerase (PDI), plays important intracellular roles during protein folding, maintaining cellular function and viability. Recent studies suggest novel roles for extracellular cell surface PDI in enhancing cellular activation and promoting their function. Moreover, a number of food-derived substances have been shown to regulate cellular PDI activity and alter disease progression. We hypothesized that PDI may have similar roles during mast cell-mediated allergic responses and examined its effects on IgE-induced mast cell activity during cell culture and food allergy. Mast cells were activated via IgE and antigen and the effects of PDI inhibition on mast cell activation were assessed. The effects of PDI blockade in vivo were examined by treating mice with the irreversible PDI inhibitor, PACMA-31, in an ovalbumin-induced model of food allergy. The role of dietary PDI modulators was investigated using various dietary compounds including curcumin and quercetin-3-rutinoside (rutin). PDI expression was observed on resting mast cell surfaces, intracellularly, and in the intestines of allergic mice. Furthermore, enhanced secretion of extracellular PDI was observed on mast cell membranes during IgE and antigen activation. Insulin turbidimetric assays demonstrated that curcumin is a potent PDI inhibitor and pre-treatment of mast cells with curcumin or established PDI inhibitors such as bacitracin, rutin or PACMA-31, resulted in the suppression of IgE-mediated activation and the secretion of various cytokines. This was accompanied by decreased mast cell proliferation, FcεRI expression, and mast cell degranulation. Similarly, treatment of allergic BALB/c mice with PACMA-31 attenuated the development of food allergy resulting in decreased allergic diarrhea, mast cell activation, and fewer intestinal mast cells. The production of TH2-specific cytokines was also suppressed. Our observations suggest that PDI catalytic activity is essential in the regulation of mast cell activation, and that its blockade may benefit patients with allergic inflammation.

Endoplasmic Reticulum Protein Disulfide Isomerase Shapes T Cell Efficacy for Adoptive Cellular Therapy of Tumors.

Effective cancer therapies simultaneously restrict tumor cell growth and improve anti-tumor immune responses. Targeting redox-dependent protein folding enzymes within the endoplasmic reticulum (ER) is an alternative approach to activation of the unfolded protein response (UPR) and a novel therapeutic platform to induce malignant cell death. E64FC26 is a recently identified protein disulfide isomerase (PDI) inhibitor that activates the UPR, oxidative stress, and apoptosis in tumor cells, but not normal cell types. Given that targeting cellular redox homeostasis is a strategy to augment T cell tumor control, we tested the effect of E64FC26 on healthy and oncogenic T cells. In stark contrast to the pro-UPR and pro-death effects we observed in malignant T cells, we found that E64FC26 improved viability and limited the UPR in healthy T cells. E64FC26 treatment also diminished oxidative stress and decreased global PDI expression in normal T cells. Oxidative stress and cell death are limited in memory T cells and we found that PDI inhibition promoted memory traits and reshaped T cell metabolism. Using adoptive transfer of tumor antigen-specific CD8 T cells, we demonstrate that T cells activated and expanded in the presence of E64FC26 control tumor growth better than vehicle-matched controls. Our data indicate that PDI inhibitors are a new class of drug that may dually inhibit tumor cell growth and improve T cell tumor control.

Activation of the Unfolded Protein Response via Inhibition of Protein Disulfide Isomerase Decreases the Capacity for DNA Repair to Sensitize Glioblastoma to Radiotherapy.

Patients with glioblastoma multiforme (GBM) survive on average 12 to 14 months after diagnosis despite surgical resection followed by radiotheraphy and temozolomide therapy. Intrinsic or acquired resistance to chemo- and radiotherapy is common and contributes to a high rate of recurrence. To investigate the therapeutic potential of protein disulfide isomerase (PDI) as a target to overcome resistance to chemoradiation, we developed a GBM tumor model wherein conditional genetic ablation of prolyl 4-hydroxylase subunit beta (P4HB), the gene that encodes PDI, can be accomplished. Loss of PDI expression induced the unfolded protein response (UPR) and decreased cell survival in two independent GBM models. Nascent RNA Bru-seq analysis of PDI-depleted cells revealed a decrease in transcription of genes involved in DNA repair and cell-cycle regulation. Activation of the UPR also led to a robust decrease in RAD51 protein expression as a result of its ubiquitination-mediated proteosomal degradation. Clonogenic survival assays demonstrated enhanced killing of GBM cells in response to a combination of PDI knockdown and ionizing radiation (IR) compared with either modality alone, which correlated with a decreased capacity to repair IR-induced DNA damage. Synergistic tumor control was also observed with the combination of PDI inhibition and IR in a mouse xenograft model compared with either single agent alone. These findings provide a strong rationale for the development of PDI inhibitors and their use in combination with DNA damage-inducing, standard-of-care therapies such as IR. SIGNIFICANCE: These findings identify PDIA1 as a therapeutic target in GBM by demonstrating efficacy of its inhibition in combination with radiotherapy through a novel mechanism involving downregulation of DNA repair genes.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2923/F1.large.jpg.

Protein disulfide isomerase as a prosurvival factor in cell therapy for muscular and vascular diseases

BackgroundCell therapy for degenerative diseases aims at rescuing tissue damage by delivery of precursor cells. Thus far, this strategy has been mostly unsuccessful due to massive loss of donor cells shortly after transplantation. Several strategies have been applied to increase transplanted cell survival but only with limited success. The endoplasmic reticulum (ER) is an organelle involved in protein folding, calcium homeostasis, and lipid biosynthesis. Protein disulfide isomerase (PDI) is a molecular chaperone induced and activated by ER stress. PDI is induced by hypoxia in neuronal, cardiac, and endothelial cells, supporting increased cell survival to hypoxic stress and protection from apoptosis in response to ischemia.MethodsWe achieved ex vivo PDI gene transfer into luciferase-expressing myoblasts and endothelial cells. We assessed cell engraftment upon intramuscular transplantation into a mouse model of Duchenne muscular dystrophy (mdx mouse) and into a mouse model of ischemic disease.ResultsWe observed that loss of full-length dystrophin expression in mdx mice muscle leads to an increase of PDI expression, possibly in response to augmented ER protein folding load. Moreover, we determined that overexpression of PDI confers a survival advantage for muscle cells in vitro and in vivo to human myoblasts injected into murine dystrophic muscle and to endothelial cells administered upon hindlimb ischemia damage, improving the therapeutic outcome of the cell therapy treatment.ConclusionsCollectively, these results suggest that overexpression of PDI may protect transplanted cells from hypoxia and other possibly occurring ER stresses, and consequently enhance their regenerative properties.

Combined expression of protein disulfide isomerase and endoplasmic reticulum oxidoreductin 1-α is a poor prognostic marker for non-small cell lung cancer.

Protein disulfide isomerase (PDI) is one of the most abundant proteins in the endoplasmic reticulum (ER) and is known as a primary ER resident target of cigarette smoke-induced oxidation. PDI dysfunction triggers unfolded protein response and ER stress. Endoplasmic reticulum oxidoreductin 1-α (ERO1A) is a major regulator of PDI, and recent evidence implicates PDI and ERO1A as tumor prognostic factors. However, the associated role of PDI and ERO1A and their prognostic impact in non-small cell lung cancers (NSCLCs) remains unknown. The present study investigated the expression of PDI and ERO1A using immunohistochemistry and examined its association with smoking status and their prognostic impact in 198 NSCLCs. PDI and ERO1A expression were observed in 71.2 and 69.2% of NSCLCs, respectively, and their expressions were significantly associated with each other (P<0.001). Individual PDI (P=0.001) and ERO1A (P=0.005) expression were significantly associated with shorter overall survival (OS) in univariate analysis. PDI expression was significantly associated with never smoking (P=0.003). PDI expression (P<0.001) and the co-expression of PDI and ERO1A (P<0.001) were independent poor prognostic factors for OS in patients with NSCLC in multivariate analysis. Individual expression and co-expression of PDI and ERO1A may be used as novel prognostic indicators of NSCLC outcome.

Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally, processes with the optimized media maintained an oxidizing intracellular environment, important for correct disulfide bond pairing, which likely contributed to reduced aggregate formation. These findings shed important understanding into how cells respond to process changes and can be useful to guide future development efforts to enhance productivity and improve product quality.

Expression of protein disulfide isomerase family members correlates with tumor progression and patient survival in ovarian cancer

ObjectiveProtein disulfide isomerase (PDI) is an oxidoreductase that is overexpressed in several cancers. PDI family members (PDIs) play a role in various diseases including cancer. Select PDIs were reported as useful markers in other cancers but their expression in ovarian cancer has not been thoroughly assessed. We sought to evaluate the expression of PDI, PDIA6, PDIR, ERp57, ERp72 and AGR3 in ovarian cancer patient samples and examine their prognostic significance.MethodsTMA samples from 415 tissues collected from three cancer centers (UM, USC, and KCCRI) were used to assess the expression levels of PDI family proteins using IHC.ResultsWe observed significant increases in PDI (p = 9.16E-36), PDIA6 (p = 5.51E-33), PDIR (p = 1.81E-12), ERp57 (p = 9.13E-07), ERp72 (p = 3.65E-22), and AGR3 (p = 4.56E-24) expression in ovarian cancers compared to normal tissues. Expression of PDI family members also increases during disease progression (p <0.001). All PDI family members are overexpressed in serous ovarian cancer (p<0.001). However, PDI, PDIA6, PDIR, ERp72 and AGR3 are more significantly overexpressed (p<0.001) than ERp57 (p<0.05) in clear cell ovarian carcinoma. Importantly, overexpression of PDI family members is associated with poor survival in ovarian cancer (p = 0.045 for PDI, p = 0.047 for PDIR, p = 0.037 for ERp57, p = 0.046 for ERp72, p = 0.040 for AGR3) with the exception of PDIA6 (p = 0.381).ConclusionsOur findings demonstrate that select PDI family members (PDI, PDIR, ERp72, ERp57 and AGR3) are potential prognostic markers for ovarian cancer.

Galectin-9 binds to O-glycans on protein disulfide isomerase.

Changes in the T cell surface redox environment regulate critical cell functions, such as cell migration, viral entry and cytokine production. Cell surface protein disulfide isomerase (PDI) contributes to the regulation of T cell surface redox status. Cell surface PDI can be released into the extracellular milieu or can be internalized by T cells. We have found that galectin-9, a soluble lectin expressed by T cells, endothelial cells and dendritic cells, binds to and retains PDI on the cell surface. While endogenous galectin-9 is not required for basal cell surface PDI expression, exogenous galectin-9 mediated retention of cell surface PDI shifted the disulfide/thiol equilibrium on the T cell surface. O-glycans on PDI are required for galectin-9 binding, and PDI recognition appears to be specific for galectin-9, as galectin-1 and galectin-3 do not bind PDI. Galectin-9 is widely expressed by immune and endothelial cells in inflamed tissues, suggesting that T cells would be exposed to abundant galectin-9, in cis and in trans, in infectious or autoimmune conditions.

Protein disulfide isomerase inhibition synergistically enhances the efficacy of sorafenib for hepatocellular carcinoma.

These results suggest that PDI is a promising therapeutic target for enhancing the efficacy of sorafenib and can also be a biomarker for predicting sorafenib responsiveness. (Hepatology 2017;66:855-868).

Protein disulfide isomerase-mediated apoptosis and proliferation of vascular smooth muscle cells induced by mechanical stress and advanced glycosylation end products result in diabetic mouse vein graft atherosclerosis

Protein disulfide isomerase (PDI) involves cell survival and death. Whether PDI mediates mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs) -triggered simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) is unknown. Here, we hypothesized that different expression levels of PDI trigger completely opposite cell fates among the different VSMC subtypes. Mouse veins were grafted into carotid arteries of non-diabetic and diabetic mice for 8 weeks; the grafted veins underwent simultaneous increases in proliferation and apoptosis, which triggered vein graft arterializations in non-diabetic or atherosclerosis in diabetic mice. A higher rate of proliferation and apoptosis was seen in the diabetic group. SS and/or AGEs stimulated the quiescent cultured VSMCs, resulting in simultaneous increases in proliferation and apoptosis; they could induce increased PDI activation and expression. Both in vivo and in vitro, the proliferating VSMCs indicated weak co-expression of PDI and SM-α-actin while apoptotic or dead cells showed strong co-expression of both. Either SS or AGEs rapidly upregulated the expression of PDI, NOX1 and ROS, and their combination had synergistic effects. Inhibiting PDI simultaneously suppressed the proliferation and apoptosis of VSMCs, while inhibition of SM-α-actin with cytochalasin D led to increased apoptosis and cleaved caspases-3 but had no effect on proliferation. In conclusion, different expression levels of PDI in VSMCs induced by SS and/or AGEs triggered a simultaneous increase in proliferation and apoptosis, accelerated vein graft arterializations or atherosclerosis, leading us to propose PDI as a novel target for the treatment of vascular remodeling and diseases.

Patagonfibrase modifies protein expression of tissue factor and protein disulfide isomerase in rat skin

Patagonfibrase is a hemorrhagic metalloproteinase isolated from the venom of the South American rear-fanged snake Philodryas patagoniensis, and is an important contributor to local lesions inflicted by this species. The tissue factor (TF)-factor VIIa complex, besides triggering the coagulation cascade, has been demonstrated to be involved in inflammatory events. Our aim was to determine whether patagonfibrase affects the expression of TF and protein disulfide isomerase (PDI), an enzyme that controls TF biological activity, at the site of patagonfibrase injection, and thus if they may play a role in hemostatic and inflammatory events induced by snake venoms. Patagonfibrase (60 μg/kg) was administered s.c. to rats, and after 3 h blood was collected to evaluate hemostasis parameters, and skin fragments close to the site of injection were taken to assess TF and PDI expression. Patagonfibrase did not alter blood cell counts, plasma fibrinogen levels, or levels of TF activity in plasma. However, by semiquantitative Western blotting, patagonfibrase increased TF expression by 2-fold, and decreased PDI expression by 3-fold in skin samples. In agreement, by immunohistochemical analyses, prominent TF expression was observed in the subcutaneous tissue. Thus, patagonfibrase affects the local expression of TF and PDI without inducing any systemic hemostatic disturbance, although that they may be involved in the local inflammatory events induced by hemorrhagic metalloproteinases. Once antivenom therapy is not totally effective to treat the local injury induced by snake venoms, modulation of the activity and expression of TF and/or PDI might become a strategy for treating snake envenomation.

Histones Induce the Procoagulant Phenotype of Endothelial Cells through Tissue Factor Up-Regulation and Thrombomodulin Down-Regulation.

The high circulating levels of histones found in various thrombotic diseases may compromise the anticoagulant barrier of endothelial cells. We determined how histones affect endothelial procoagulant tissue factor (TF) and anticoagulant thrombomodulin (TM). Surface antigens, soluble forms, and mRNA levels of TF and TM were measured by flow cytometry, ELISA, and real-time RT-PCR, respectively. TF and TM activity were measured using procoagulant activity, thrombin generation, or chromogenic assays. Involvement of the toll-like receptor (TLR) was assessed using the neutralizing antibodies. Histones dose-dependently induced surface antigens, activity and mRNA levels of endothelial TF. Histone-treated endothelial cells significantly shortened the lag time and enhanced the endogenous thrombin potential of normal plasma, which was normalized by a TF neutralizing antibody. Histones induced phosphatidylserine and protein-disulfide isomerase expression in endothelial cells. Histones also reduced the surface antigen, activity, and mRNA levels of endothelial TM. Polysialic acid and heparin reversed the histone-induced TF up-regulation and TM down-regulation. Activated protein C did not affect the TF up-regulation, but interrupted TM down-regulation. TLR2, and TLR4 inhibitors partially blocked the TF up-regulation. Histones induced the endothelial procoagulant phenotype through TF up-regulation and TM down-regulation. The effects of histones were partly mediated by TLR2, TLR4. Strategies to inhibit the harmful effects of histones in endothelial cells may be required in order to prevent a thrombotic environment.

The Effect of Protein Disulfide Isomerase Expression and Activity on Disulfide Bond Formation in Wheat Grains during Ripening

The Effect of Protein Disulfide Isomerase Expression and Activity on Disulfide Bond Formation in Wheat Grains during Ripening

PP38 - PDIA1 overexpression activates acutely Nox1 NADPH oxidase in VSMC

Mechanisms regulating NADPH oxidase remain open and include the redox chaperone protein disulfide isomerase (PDI). We described previously that PDI transient overexpression in VSMC enhances basal Nox1 mRNA expression and activity, without any further response for AngII stimulus in ROS production. Here we investigated whether Nox1 activation mediated by PDI overexpression may involve AngII receptor (AT1R).We developed rabbit aortic VSMC with doxicycline inducible system for rat PDI (VSMC-PDI), which showed 3,5 fold increased total PDI expression after 48h of doxicycline addition. VSMC-PDI showed increased superoxide production by NADPH-triggered enriched membrane fraction (1 vs. 2,5 ± 0,03 RLU/mg protein, lucigenin reduction), which was totally inhibited by Nox1/4 inhibitor (GKT136901, 100uM). VSMC-PDI showed higher migration trajectories vs. VSMC with no stimulus (1 vs. 1,75 ± 0,04 pixels, end distance from origin by single cell migration assay for 16h). Although inhibitors of AT1R (losartan and candesartan, 10-100nM for 16h) decreased intracellular superoxide (2-hydroxyethidium quantification by HPLC) or extracellular H2O2 (AmplexRed assay) productions mediated by PDI transient overexpression, there is no direct PDI interaction with AT1R, based on several confocal microscopy analyzes in both VSMC and HEK293 cells transfected with AT1R-GFP. Since AT1R activates β-arrestin1/2 signaling, we silenced β-arrestin1/2 with further PDI induction in VSMC-PDI. Preliminary results suggest no changes in Nox1 NADPH activity. Finally, chronic VSMC-PDI (obtained after antibiotic selection of VSMC transiently transfected, 4 fold increased PDI expression) showed no more increased intracellular superoxide or Nox1 NADPH oxidase activation compared to basal VSMC. The mechanisms underlying Nox1 acute activation by PDI overexpression in VSMC does not involve direct interaction with AT1R, and might not involve β-arrestin1/2. Chronic PDI overexpression may induce VSMC redox adaptations, since Nox1 NADPH oxidase activity is decayed to basal levels.

Glycated human serum albumin isolated from poorly controlled diabetic patients impairs cholesterol efflux from macrophages: an investigation by mass spectrometry.

Advanced glycation end-products impair ABCA-1-mediated cholesterol efflux by eliciting inflammation, the generation of reactive oxygen species and endoplasmatic reticulum (ER) stress. The glycation level of human serum albumin (HSA) from type 1 and type 2 diabetic patients was determined by matrix assisted laser desorption/ionization (MALDI) mass spectrometry and related to possible impairment of ER function and cellular cholesterol efflux. Comparison of the MALDI spectra from healthy and diabetic subjects allowed us to determine an increased HSA mean mass of 1297 Da for type 1 and 890 Da for type 2. These values reflect a mean condensation of at least 8 glucose units and 5 glucose units, respectively. Mouse peritoneal macrophages were treated with HSA from control, type 1 and type 2 diabetic subjects in order to measure the expression of Grp78, Grp94, protein disulfide isomerase (PDI), calreticulin (CRT) and ABCA-1. (14)C-cholesterol overloaded-J774 macrophages were treated with HSA from control and diabetic subjects and further incubated with apo A-1 to determine the cholesterol efflux. Combined analyses comprising HSA from type 1 and type 2 diabetic patients were performed in cellular functional assays. In macrophages, PDI expression increased 89% and CRT 3.4 times in comparison to HSA from the control subjects. ABCA-1 protein level and apo A-I mediated cholesterol efflux were, respectively, 50% and 60% reduced in macrophages exposed to HSA from type 1 and type 2 diabetic patients when compared to that exposed to HSA from control subjects. We provide evidence that the level of glycation that occurs in albumin in vivo damages the ER function related to the impairment in macrophage reverse cholesterol transport and so contributes to atherosclerosis in diabetes.