Abstract 17269: Extracelllular Protein Disulfide Isomerase Reshapes Vascular Architecture Against Constrictive Remodeling

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Haniel A Araujo ◽  
Leonardo Y Tanaka ◽  
Gustavo K Hironaka ◽  
Thais L Araujo ◽  
Celso K Takimura ◽  
...  
Keyword(s):  
Vascular Remodeling ◽  
Protein Disulfide Isomerase ◽  
Fiber Type ◽  
Vascular Diseases ◽  
Collagen Type I ◽  
Type I ◽  
Disulfide Isomerase ◽  
Protein Disulfide

INTRODUCTION: Vascular remodeling orchestrates a complex network of signaling pathways responsible for pathological changes in many vascular diseases such as atherosclerosis. We investigated the role of endoplasmic reticulum chaperone Protein Disulfide Isomerase (PDI) and the extracellular PDI (ecPDI) pool in vascular caliber and architecture during vascular repair and remodeling after injury (AI). METHODS AND RESULTS: After rabbit iliac artery balloon injury, PDI is markedly increased at mRNA and protein levels (25-fold vs. basal 14 days AI), with increase in both intracellular and ecPDI. Silencing PDI by siRNA in vitro induced ER stress markers upregulation and apoptosis (assessed by TUNEL assay). PDI knockdown also upregulated proliferation marker PCNA and decreased differentiation marker calponin-C. Furthermore, ecPDI inhibition prevents injury-increased hydrogen peroxide generation and decreases arterial nitrate (NO3-) level. EcPDI neutralization in vivo with PDIAb-containing perivascular gel from days 12-14AI promoted 25% decrease in vascular caliber at arteriography and similar decreases in total vessel circumference at optical coherence tomography, without changing neointima, indicating increased constrictive remodeling. EcPDI neutralization promoted striking changes in collagen, with switch from circumferential to radial fiber orientation towards a more rigid fiber type. Collagen type I and III were decreased after ecPDI inhibition in arteries 14 days AI. Cytoskeleton architecture was also disrupted, with loss of stress fiber coherent organization and switch from thin to medium-thickness actin fibers. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling. There was decreased PDI expression in media and intima from plaques exhibiting constrictive remodeling and, conversely, enhanced PDI expression in media of plaques depicting outward remodeling. CONCLUSIONS: Thus, PDI is highly upregulated after injury and reshapes matrix and cytoskeleton architecture to support an anticonstrictive remodeling effect. Such findings suggest an important role for PDI in lumen maintenance during vascular remodeling.

Abstract 806: Protein Disulfide Isomerase (PDI) signals Tissue Factor (TF)-dependent Fibrin formation in vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Marie-Luise von Bruehl ◽  
Lenka Grahl ◽  
Ildiko Konrad ◽  
Michael Lorenz ◽  
Christian Schulz ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Arterial Thrombosis ◽  
Protein Disulfide Isomerase ◽  
Disulfide Isomerase ◽  
Intravascular Coagulation ◽  
Disulfide Formation ◽  
Fibrin Formation ◽  
Protein Disulfide

Background: Pathological fibrin formation is a predominant cause for arterial thrombosis, triggering myocardial infarction and stroke. Tissue factor (TF), which is expressed by vascular endothelium and cell-derived microparticles, is a central trigger of intravascular fibrin generation. In intact blood vessels, TF is only minimally active, and the coagulation system is down-regulated. In contrast, TF is rapidly stimulated following endothelial disruption. However, the molecular mechanisms that trigger fibrin formation via regulation of TF activation are largely undefined. Methods and Results: Here, we have identified a novel pathway that leads to TF activation in vitro and contributes to intravascular coagulation in vivo. We show that the protein disulfide isomerase (PDI) induces disulfide formation of the Cys186/Cys209 pair located in the extracellular domain of TF. In vitro, PDI-mediated disulfide formation led to profound activation of TF. In vivo, we found that PDI is not present in the intact vessel wall, but becomes abundantly expressed following vessel damage. We show that injured smooth muscle cells and adherent/activated platelets are the major cellular sources of PDI at sites of vascular injury. We then assessed fibrin formation following injury of the common carotid artery using intravital microscopy. We observed that PDI-blockade strongly reduced fibrin formation by approximately 45– 60%. Likewise, intravenous infusion of PDI significantly enhanced TF-dependent fibrin formation. Together, this implies that PDI initiates coagulation in vitro and also during arterial thrombosis in vivo. Notably, PDI did not act on platelets to trigger intravascular coagulation. Correspondingly, PDI blockade also inhibited fibrin formation, when platelet adhesion was prevented using a function-blocking anti-GPVI antibody. Conclusion: These findings reveal for the first time in vivo that the thiol isomerase PDI is a major molecular trigger of blood coagulation, which is exposed after vessel injury and converts TF into its functionally active form. Hence, PDI represents a promising novel target for the treatment of pathologic fibrin formation in patients with thrombotic disorders.

scholarly journals Novel anti-thrombotic agent for modulation of protein disulfide isomerase family member ERp57 for prophylactic therapy

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Guozhen Cui ◽  
Luchen Shan ◽  
Lin Guo ◽  
Ivan Keung Chu ◽  
Guohui Li ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Protein Disulfide Isomerase ◽  
Thrombus Formation ◽  
Myocardial Infarct ◽  
Redox Activity ◽  
Acute Myocardial Infarct ◽  
Disulfide Isomerase ◽  
Protein Disulfide

Abstract Protein disulfide isomerase (PDI) family members including PDI and ERp57 emerge as novel targets for anti-thrombotic treatments, but chemical agents with selectivity remain to be explored. We previously reported a novel derivative of danshensu (DSS), known as ADTM, displayed strong cardioprotective effects against oxidative stress-induced cellular injury in vitro and acute myocardial infarct in vivo. Herein, using chemical proteomics approach, we identified ERp57 as a major target of ADTM. ADTM displayed potent inhibitory effects on the redox activity of ERp57, inhibited the adenosine diphosphate (ADP)-induced expressions of P-selectin and αIIbβ3 integrin and disrupted the interaction between ERp57 and αIIbβ3. In addition, ADTM inhibited both arachidonic acid (AA)-induced and ADP-induced platelet aggregation in vitro. Furthermore, ADTM significantly inhibited rat platelet aggregation and thrombus formation in vivo. Taken together, ADTM represents a promising candidate for anti-thrombotic therapy targeting ERp57.

scholarly journals Collagen-Based Electrospun Materials for Tissue Engineering: A Systematic Review

2021 ◽  
Vol 8 (3) ◽  
pp. 39
Author(s):  
Britani N. Blackstone ◽  
Summer C. Gallentine ◽  
Heather M. Powell
Keyword(s):  
Systematic Review ◽  
Tissue Engineering ◽  
Collagen Type I ◽  
The Body ◽  
Pool Data ◽  
Type I ◽  
High Concentrations ◽  
Increased Strength

Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.

scholarly journals Plant Recombinant Human Collagen Type I Hydrogels for Corneal Regeneration

2021 ◽  
Author(s):  
Michel Haagdorens ◽  
Elle Edin ◽  
Per Fagerholm ◽  
Marc Groleau ◽  
Zvi Shtein ◽  
...  
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Collagen Fibrils ◽  
Type I ◽  
Safe Alternative ◽  
Human Donor ◽  
Human Collagen ◽  
Donor Corneas

Abstract Purpose To determine feasibility of plant-derived recombinant human collagen type I (RHCI) for use in corneal regenerative implants Methods RHCI was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogels. Application of shear force to liquid crystalline RHCI aligned the collagen fibrils. Both aligned and random hydrogels were evaluated for mechanical and optical properties, as well as in vitro biocompatibility. Further evaluation was performed in vivo by subcutaneous implantation in rats and corneal implantation in Göttingen minipigs. Results Spontaneous crosslinking of randomly aligned RHCI (rRHCI) formed robust, transparent hydrogels that were sufficient for implantation. Aligning the RHCI (aRHCI) resulted in thicker collagen fibrils forming an opaque hydrogel with insufficient transverse mechanical strength for surgical manipulation. rRHCI showed minimal inflammation when implanted subcutaneously in rats. The corneal implants in minipigs showed that rRHCI hydrogels promoted regeneration of corneal epithelium, stroma, and nerves; some myofibroblasts were seen in the regenerated neo-corneas. Conclusion Plant-derived RHCI was used to fabricate a hydrogel that is transparent, mechanically stable, and biocompatible when grafted as corneal implants in minipigs. Plant-derived collagen is determined to be a safe alternative to allografts, animal collagens, or yeast-derived recombinant human collagen for tissue engineering applications. The main advantage is that unlike donor corneas or yeast-produced collagen, the RHCI supply is potentially unlimited due to the high yields of this production method. Lay Summary A severe shortage of human-donor corneas for transplantation has led scientists to develop synthetic alternatives. Here, recombinant human collagen type I made of tobacco plants through genetic engineering was tested for use in making corneal implants. We made strong, transparent hydrogels that were tested by implanting subcutaneously in rats and in the corneas of minipigs. We showed that the plant collagen was biocompatible and was able to stably regenerate the corneas of minipigs comparable to yeast-produced recombinant collagen that we previously tested in clinical trials. The advantage of the plant collagen is that the supply is potentially limitless.

scholarly journals Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Audrey Beaussart ◽  
Tristan Brandhorst ◽  
Yves F. Dufrêne ◽  
Bruce S. Klein
Keyword(s):  
Protein Disulfide Isomerase ◽  
Tandem Repeats ◽  
Pathogenic Fungi ◽  
Heparin Binding ◽  
Binding Studies ◽  
Disulfide Linkage ◽  
Disulfide Isomerase ◽  
Atomic Force ◽  
Protein Disulfide

ABSTRACT Blastomyces adhesin-1 (BAD-1) protein mediates the virulence of the yeast Blastomyces dermatitidis, in part by binding host lung tissue, the extracellular matrix, and cellular receptors via glycosaminoglycans (GAGs), such as heparan sulfate. The tandem repeats that make up over 90% of BAD-1 appear in their native state to be tightly folded into an inactive conformation, but recent work has shown that they become activated and adhesive upon reduction of a disulfide linkage. Here, atomic force microscopy (AFM) of a single BAD-1 molecule interacting with immobilized heparin revealed that binding is enhanced upon treatment with protein disulfide isomerase and dithiothreitol (PDI/DTT). PDI/DTT treatment of BAD-1 induced a plateau effect in atomic force signatures that was consistent with sequential rupture of tandem binding domains. Inhibition of PDI in murine macrophages blunted BAD-1 binding to heparin in vitro. Based on AFM, we found that a short Cardin-Weintraub sequence paired with a WxxWxxW sequence in the first, degenerate repeat at the N terminus of BAD-1 was sufficient to initiate heparin binding. Removal of half of the 41 BAD-1 tandem repeats led to weaker adhesion, illustrating their role in enhanced binding. Mass spectroscopy of the tandem repeat revealed that the PDI-induced interaction with heparin is characterized by ruptured disulfide bonds and that cysteine thiols remain reduced. Further binding studies showed direct involvement of thiols in heparin ligation. Thus, we propose that the N-terminal domain of BAD-1 governs the initial association with host GAGs and that proximity to GAG-associated host PDI catalyzes activation of additional binding motifs conserved within the tandem repeats, leading to enhanced avidity and availability of reduced thiols. IMPORTANCE Pathogenic fungi and other microbes must adhere to host tissue to initiate infection. Surface adhesins promote this event and may be required for disease pathogenesis. We studied a fungal adhesin essential for virulence (BAD-1; Blastomyces adhesin-1) and found that host products induce its structural reconfiguration and foster its optimal binding to tissue structures.

scholarly journals WNT-5A regulates TGF-β-related activities in liver fibrosis

2017 ◽  
Vol 312 (3) ◽  
pp. G219-G227 ◽  
Author(s):  
Leonie Beljaars ◽  
Sara Daliri ◽  
Christa Dijkhuizen ◽  
Klaas Poelstra ◽  
Reinoud Gosens
Keyword(s):  
Liver Fibrosis ◽  
Functional Role ◽  
Collagen Type ◽  
Collagen Type I ◽  
Matrix Proteins ◽  
Type I ◽  
Human Livers

WNT-5A is a secreted growth factor that belongs to the noncanonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines, and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared with healthy livers. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa colocalizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and proinflammatory cytokines IL-1β and TNF-α. Additionally, TGF-β induces the expression of the WNT receptors FZD2 and FZD8. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured compared with nonsilencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A in vitro and in vivo. IFNγ-treated fibrotic mice showed significantly less WNT-5A expression compared with untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role in TGF-β-induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo. NEW & NOTEWORTHY This study describes the localization and functional role of WNT-5A in human and mouse fibrotic livers. Hepatic WNT-5A expression parallels collagen type I expression. In vivo and in vitro, the myofibroblasts were identified as the key hepatic cells producing WNT-5A. WNT-5A is under control of TGF-β and its activities are primarily profibrotic.

Protein Disulfide Isomerase-A1 Overexpression Attenuates Vascular Calcification in Vivo

2020 ◽  
Vol 159 ◽  
pp. S95
Author(s):  
Luciana Pescatore ◽  
Patricia Nolasco ◽  
Melissa Fessel ◽  
Youri Almeida ◽  
João Wosniak ◽  
...  
Keyword(s):  
Vascular Calcification ◽  
Protein Disulfide Isomerase ◽  
Disulfide Isomerase ◽  
Protein Disulfide

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.

Protein Disulfide Isomerase Regulates Sickle Erythrocyte Volume Via ET-1 Dependent Casein Kinase II Mechanism

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2114-2114
Author(s):  
Shirley D Valentin-Berrios ◽  
Jose R Romero ◽  
Alicia Rivera
Keyword(s):  
Plasma Membrane ◽  
Sickle Cell ◽  
Protein Disulfide Isomerase ◽  
Disulfide Isomerase ◽  
Sickle Erythrocyte ◽  
Cellular Dehydration ◽  
Sickle Erythrocytes ◽  
Protein Disulfide

Abstract Abstract 2114 Disordered K+ efflux and osmotically induced water loss leads to red blood cell (RBC) dehydration and plays a role in the pathophysiology of Sickle Cell Disease. We previously reported that activation of endothelin-1 (ET-1) receptors in sickle erythrocyte was partially responsible for dense sickle cell formation. However, the mechanism by which ET-1 regulates RBC volume remains unclear. Serine/threonine kinases have been shown to regulate K+ transport in RBC. Casein Kinase II (CK2), a serine/threonine kinase, phosphorylates acidic proteins, regulates calmodulin activity and cytoskeletal proteins and is present in RBC. CK2 activity is blocked by apigenin, emodin, heparin, and ornithine decarboxylase. Previous reports have shown a role for flavonoids such as apigenin as substrates for erythrocyte plasma membrane oxidoreductases. We recently observed a role for Protein Disulfide Isomerase (PDI) in regulating cellular hydration and K+ efflux in human RBC. PDI catalyzes disulfide interchange reactions in the plasma membrane, mediates redox modifications and is up-regulated under hypoxic conditions. However the relationship between CK2 and PDI in the setting of cellular hydration status is un-explored. Our results indicate that erythrocyte membrane CK2 activity increases when sickle cells are incubated with 500 nM ET-1 for 30 min (2.8 ± 0.1 to 4.9 ± 0.01 nmol/min/mL * 106 cell) an event that is blunted by pre-incubation with the ET-1 B receptor blocker, BQ788 (2.5 ± 0.1 nmol/min/mL * 106 cell, n=3, p<0.04) and 20 μM apigenin (2.7 ± 0.4 nmol/min/mL * 106 cell, n=3, p<0.04). We examined the role of CK2 activation on cellular dehydration. We incubated sickle erythrocytes for 3 hours in deoxygenation-oxygenation cycles in the presence or absence of 20μM apigenin or 2μM 4,5,6,7-tetrabromobenzotriazole (TBB), a specific CK2 inhibitor, and measured the changes in erythrocyte density by phthalate oil density analysis. We observed that inhibition of CK2 led to reduced deoxygenation-stimulated cellular dehydration in sickle erythrocytes by apigenin (D50= 1.106 to 1.100 g/mL) or TBB (D50 =1.097 g/mL). We then studied the role of CK2 inhibitors on PDI activity by Insulin Turbidity Assay and observed that apigenin and TBB led to significant reductions in PDI activity in vitro (64% and 42% respectively). We also studied the effects of the flavonoids: naringenin, naringin, apigenin and rutin on PDI activity and observed reductions in PDI activity that were greater with apigenin>rutin>TBB>naringin>naringenin (n=2, P<0.05). Furthermore, we observed that K+ flux via Gardos channel activation is correlated with PDI activity in vitro in sickle erythrocytes. Taken together our results implicate CK2 and PDI as intermediate regulators of ET-1 stimulated cellular volume systems in red blood cells. Supported by NIH R01-HL09632 to AR. Disclosures: No relevant conflicts of interest to declare.

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