JAM-A Acts via C/EBP-α to Promote Claudin-5 Expression and Enhance Endothelial Barrier Function

Rationale: Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive. Objective: We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions. Methods and Results: Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of β-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A–C/EBP-α–mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer. Conclusions: We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction. Graphic Abstract: An graphic abstract is available for this article.

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A collagen gel-coated, aligned nanofiber membrane for enhanced endothelial barrier function

Scientific Reports ◽

10.1038/s41598-019-51560-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Dohui Kim ◽

Seongsu Eom ◽

Sang Min Park ◽

Hyeonjun Hong ◽

Dong Sung Kim

Keyword(s):

Tight Junctions ◽

Barrier Function ◽

Synergistic Effects ◽

Umbilical Vein ◽

Collagen Gel ◽

Electrospinning Process ◽

Endothelial Barrier ◽

Nanofiber Membrane ◽

Endothelial Barrier Function ◽

Barrier Dysfunction

Abstract Herein, a collagen gel-coated and aligned nanofiber membrane named Col-ANM is developed, which remarkably improves endothelial barrier function by providing biochemical and topographical cues simultaneously. Col-ANM is fabricated by collagen gel coating process on an aligned polycaprolactone (PCL) nanofiber membrane, which is obtained by a simple electrospinning process adopting a parallel electrode collector. Human umbilical vein endothelial cells (HUVECs) cultured on Col-ANM exhibit remarkably enhanced endothelial barrier function with high expression levels of intercellular junction proteins of ZO-1 and VE-cadherin, a high TEER, and a cellular permeability compared with the artificial porous membranes in commercial cell culture well inserts. The enhanced endothelial barrier function is conjectured to be attributed to the synergistic effects of topographical and biochemical cues provided by the aligned PCL nanofibers and collagen gel in the Col-ANM, respectively. Finally, the reactive oxygen species is applied to the HUVEC monolayer formed on the Col-ANM to destroy the tight junctions between HUVECs. The destruction of the tight junctions is demonstrated by the decreased TEER value over time. Results indicate the potential of Col-ANM in modeling endothelial barrier dysfunction-related diseases.

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Abstract 45: Endothelial Aryl Hydrocarbon Receptor Nucleatranslator (Arnt) is a Novel Regulator of Cardiac Endothelial Barrier Integrity in Diabetes

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.45 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Maura Knapp ◽

Mei Zheng ◽

Nikola Sladojevic ◽

Qiong Zhao ◽

Konstaintin G Birukov ◽

...

Keyword(s):

Endothelial Cells ◽

Barrier Function ◽

Endothelial Permeability ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Aryl Hydrocarbon ◽

Underlying Mechanisms

Background: Diabetes leads to endothelial barrier dysfunction and altered endothelial permeability, which results in increased cardiovascular risk. ARNT, also known as HIF-1β, a transcription factor that functions as a master regulator of glucose homeostasis, has been implicated in diabetes. Endothelial-specific ARNT deletion (ArntΔEC) in mice is embryonically lethal, with hemorrhage occurring in the heart during the embryonic stage. However, the particular role of endothelial ARNT(ecARNT) in diabetes is largely unknown. We have found a significant decrease in ARNT expression in both diabetic rodent endothelial cells and diabetic human hearts. We hypothesize that a loss of ecARNT mediates endothelial barrier dysfunction during diabetes. Methods and Results: We generated inducible endothelial specific ARNT knockout mice (ecARNT-/-) by crossing mice with loxP sequences flanking exon 6 of ARNT with Cre ERT2 mice under the VE-cadherin promoter. A 90% deletion of ecARNT was achieved following two weeks of oral tamoxifen administration. ecARNT-/- mice exhibit severe blood vessel leakage, which is restricted to the heart, suggesting a distinct function for ecARNT in different tissues. Cardiomyopathy is evident 6 months after ARNT deletion. In vitro , trans-endothelial electrical resistance (TER) and transwell assays have confirmed endothelial barrier disruption in cardiac microvascular endothelial cells (CMEC) isolated from both ecARNT-/- hearts and diabetic (DB/DB) mouse hearts. To determine the underlying mechanisms by which ARNT may regulate endothelial barrier function, we performed DNA sequencing on CMEC isolated from control, ecARNT-/-, and DB/DB mice. Data suggest a significant increase in TNFa signaling, including ELAM-1 and ICAM-1 in CMEC isolated from ecARNT-/- CMEC and diabetic CMEC. Moreover, use of anti-TNFa antibody rescues endothelial barrier dysfunction in CMEC isolated from ecARNT-/- mice. Taken together, these results suggest that a reduction in ecARNT during diabetes may mediate endothelial barrier dysfunction through a TNFa signaling pathway. Conclusion: ecARNT is a critical mediator of endothelial barrier function and could potentially serve as a therapeutic target for diabetic cardiovascular diseases.

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Development of an Image-Based HCS-Compatible Method for Endothelial Barrier Function Assessment

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211030900 ◽

2021 ◽

pp. 247255522110309

Author(s):

Oleksii Dubrovskyi ◽

Erica Hasten ◽

Steven M. Dudek ◽

Michael T. Flavin ◽

Leo Li-Ying Chan

Keyword(s):

Drug Discovery ◽

Barrier Function ◽

Time Course ◽

Molecular Mechanisms ◽

Cell Monolayer ◽

Endothelial Permeability ◽

Image Cytometry ◽

Endothelial Barrier ◽

Published Data ◽

Endothelial Barrier Function

The recent renascence of phenotypic drug discovery (PDD) is catalyzed by its ability to identify first-in-class drugs and deliver results when the exact molecular mechanism is partially obscure. Acute respiratory distress syndrome (ARDS) is a severe, life-threatening condition with a high mortality rate that has increased in frequency due to the COVID-19 pandemic. Despite decades of laboratory and clinical study, no efficient pharmacological therapy for ARDS has been found. An increase in endothelial permeability is the primary event in ARDS onset, causing the development of pulmonary edema that leads to respiratory failure. Currently, the detailed molecular mechanisms regulating endothelial permeability are poorly understood. Therefore, the use of the PDD approach in the search for efficient ARDS treatment can be more productive than classic target-based drug discovery (TDD), but its use requires a new cell-based assay compatible with high-throughput (HTS) and high-content (HCS) screening. Here we report the development of a new plate-based image cytometry method to measure endothelial barrier function. The incorporation of image cytometry in combination with digital image analysis substantially decreases assay variability and increases the signal window. This new method simultaneously allows for rapid measurement of cell monolayer permeability and cytological analysis. The time-course of permeability increase in human pulmonary artery endothelial cells (HPAECs) in response to the thrombin and tumor necrosis factor α treatment correlates with previously published data obtained by transendothelial resistance (TER) measurements. Furthermore, the proposed image cytometry method can be easily adapted for HTS/HCS applications.

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Flow dynamics control endothelial permeability in a microfluidic vessel bifurcation model

Lab on a Chip ◽

10.1039/c8lc00130h ◽

2018 ◽

Vol 18 (7) ◽

pp. 1084-1093 ◽

Cited By ~ 14

Author(s):

Ehsan Akbari ◽

Griffin B. Spychalski ◽

Kaushik K. Rangharajan ◽

Shaurya Prakash ◽

Jonathan W. Song

Keyword(s):

Blood Flow ◽

Barrier Function ◽

Molecular Mechanisms ◽

Endothelial Permeability ◽

Flow Dynamics ◽

Endothelial Barrier ◽

Endothelial Barrier Function

Endothelial barrier function is known to be regulated by a number of molecular mechanisms; however, the role of biomechanical signals associated with blood flow is comparatively less explored.

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Abstract 132: Endothelial Progenitor Cells Prevent Endothelial Barrier Injury by Cdc42-dependent Assembly of VE-cadherin

Circulation ◽

10.1161/circ.116.suppl_16.ii_4 ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Hiroshi Ohkawra ◽

Yidan D. Zhao ◽

Asrar B. Malik

Keyword(s):

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Barrier Function ◽

Inflammatory Diseases ◽

Vascular Inflammation ◽

Endothelial Permeability ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Endothelial Progenitor

We investigated the use of bone-marrow derived endothelial progenitor cells (EPCs) for the treatment of microvascular endothelial injury caused by inflammatory diseases. As activation of the monomeric RhoGTPase Cdc42 signals annealing of adherens junctions (AJs) in the endothelial barrier, we examined the possibility that EPCs could restore endothelial barrier dysfunction by promoting Cdc42 activation in endothelial cells (ECs). We observed that co-culture of EPCs with ECs prevented the decrease in transendothelial electrical resistance (TER), a measure of AJ assembly, and the decrease in VE-cadherin expression induced by thrombin (4 U/mL). EPCs also significantly reduced the formation of inter-endothelial gaps mediated by RhoA activation as well as myosin light chain phosphorylation in response to thrombin. We showed that EPCs induced activation of Cdc42 in ECs and increased TER within 15 min after the addition EPCs to ECs, indicating the enhancement of endothelial barrier function. Addition of EPCs after thrombin-induced disruption of AJs also promoted rapid recovery of TER compared to controls. Based on siRNA knockdown data, the endothelial barrier protective effect of EPCs was mediated by Cdc42 activation in ECs. Thus, EPC-mediated activation of Cdc42 in ECs decreases basal endothelial permeability and prevents endothelial hyper-permeability induced by the mediator thrombin. EPC/EC cross-talk is a critical mechanism regulating endothelial barrier function suggesting the value of EPCs in the treatment of microvascular injury associated with vascular inflammation.

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ID: 114: AN ANTAGONIST OF LYSOPHOSPHATIDIC ACID RECEPTOR1, AM966, INCREASES PULMONARY ENDOTHELIAL PERMEABILITY THROUGH ACTIVATION OF VE-CADHERIN

Journal of Investigative Medicine ◽

10.1136/jim-2016-000120.112 ◽

2016 ◽

Vol 64 (4) ◽

pp. 965.3-966

Author(s):

J Cai ◽

J Wei ◽

AM Jacko ◽

J Zhao

Keyword(s):

Lysophosphatidic Acid ◽

Barrier Function ◽

Endothelial Permeability ◽

Heart Association ◽

Endothelial Barrier ◽

Dependent Manner ◽

Gap Formation ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Barrier Integrity

BackgroundMaintenance of pulmonary endothelial barrier integrity is important for reducing severity of lung injury. VE-cadherin is a major component of cell–cell adherens junctions in endothelium. In response to inflammatory stimuli, VE-cadherin is tyrosine phosphorylated, resulting in dissociation with catenins, which links to f-actin. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid, which regulates cell motility. LPA has been shown to increase lung epithelial barrier integrity, while it reduces endothelial barrier function. AM966 is an antagonist exhibiting an anti-fibrotic property. However, the effect of AM966 on pulmonary endothelial barrier integrity has not been well studied.Methods and ResultsTo investigate endothelial barrier integrity, electric cell-substrate sensing (ECIS) system was used to measure permeability in human lung microvascular endothelial cells (HLMVECs). Similar to the effect of LPA, AM966 increases permeability immediately in a dose dependent manner. To investigate the molecular mechanism by which regulates AM966-mediated reduction of endothelial barrier function, HLMVECs were treated with AM966, and then phosphorylation of myosin light chain (MLC) and VE-cadherin were determined by immunoblotting. AM966 increased phosphorylation of MLC and VE-cadherin. VE-cadherin and f-actin double immunostaining revealed that AM966 induces gap formation and f-actin stress fibers as well as dissociation between VE-cadherin and f-actin.ConclusionThis study reveals that AM966 induces lung endothelial barrier dysfunction, which is regulated by phosphorylation of VE-cadherin.This work was supported by the National Institutes of Health (R01GM115389 to J.Z.), American Heart Association 12SDG9050005 (J.Z.), American Lung Association Biomedical Research Grant RG350146 (J.Z.).

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Abstract 73: Bone Morphogenetic Protein Activity Modulates Endothelial Barrier Function

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a73 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Thomas Helbing ◽

Elena Ketterer ◽

Bianca Engert ◽

Jennifer Heinke ◽

Sebastian Grundmann ◽

...

Keyword(s):

Acute Lung Injury ◽

Lung Injury ◽

Barrier Function ◽

Endothelial Permeability ◽

Bmp Signaling ◽

Endothelial Barrier ◽

Endothelial Barrier Function

Introduction: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are associated with high morbidity and mortality in patients. During the progression of ALI, the endothelial cell barrier of the pulmonary vasculature becomes compromised, leading to pulmonary edema, a characteristic feature of ALI. It is well-established that EC barrier dysfunction is initiated by cytoskeletal remodeling, which leads to disruption of cell-cell contacts and formation of paracellular gaps, allowing penetration of protein-rich fluid and inflammatory cells. Bone morphogenetic proteins (BMPs) are important players in endothelial dysfunction and inflammation but their effects on endothelial permeability in ALI have not been investigated until now. Methods and Results: As a first approach to assess the role of BMPs in acute lung injury we analysed BMP4 and BMPER expression in an infectious (LPS) and a non-infectious (bleomycin) mouse models of acute lung injury. In both models BMP4 and BMPER protein expression levels were reduced demonstrated by western blots, suggesting that BMPs are involved in progression ALI. To assess the role of BMPs on vascular leakage, a key feature of ALI, BMP activity in mice was inhibited by i.p. administration of LDN193189, a small molecule that blocks BMP signalling. After 3 days Evans blue dye (EVB) was administered i.v. and dye extravasation into the lungs was quantified as a marker for vascular leakage. Interestingly, LDN193189 significantly increased endothelial permeability compared to control lungs, indicating that BMP signaling is involved in maintenance of endothelial barrier function. To quantify effects of BMP inhibition on endothelial barrier function in vitro, HUVECs were seeded onto transwell filters and were exposed to LDN193189. After 3 days FITC-dextrane was added and passage into the lower chamber was quantified as a marker for endothelial barrier function. Thrombin served as a positive control. As expected from our in vivo experiments inhibition of BMP signaling by LDN193189 enhanced FITC-dextrane passage. To study specific effects of BMPs on endothelial barrier function, two protagonist of the BMP family, BMP2 and BMP4, or BMP modulator BMPER were tested in the transwell assay in vitro. Interestingly BMP4 and BMPER, but not BMP2, reduced FITC-dextrane passage demonstrating that BMP4 and BMPER improved endothelial barrier function. Vice versa, specific knock down of BMP4 or BMPER increased leakage in transwell assays. Im immuncytochemistry silencing of BMPER or BMP4 induced hyperpermeability as a consequence of a pro-inflammatory endothelial phenotype characterised by reduced cell-cell contacts and increased actin stress fiber formation. Additionally, the pro-inflammatory endothelial phenotype was confirmed by real-time revealing increased expression of adhesion molecules ICAM-1 or proinflammatory cytokines such as IL-6 and IL-8 in endothelial cells after BMPER or BMP4 knock down. Confirming these in vitro results BMPER +/- mice exhibit increased extravasation of EVB into the lungs, indicating that partial loss of BMPER impairs endothelial barrier function in vitro and in vivo. Conclusion: We identify BMPER and BMP4 as local regulators of vascular permeability. Both are protective for endothelial barrier function and may open new therapeutic avenues in the treatment of acute lung injury.

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Effect of p18 on Endothelial Barrier Function by Mediating Vascular Endothelial Rab11a-VE-cadherin Recycling

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab172 ◽

2021 ◽

Author(s):

Bo-Wen Xu ◽

Zhi-Qiang Cheng ◽

Xu-Ting Zhi ◽

Xiao-Mei Yang ◽

Zhi-Bo Yan

Keyword(s):

Barrier Function ◽

Adherens Junctions ◽

Cecal Ligation ◽

Underlying Mechanism ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Recycling Endosome

Abstract Endothelial barrier integrity requires recycling of VE-cadherin to adherens junctions. Both p18 and Rab11a play significant roles in VE-cadherin recycling. However, the underlying mechanism and the role of p18 in activating Rab11a have yet to be elucidated. Performing in vitro and in vivo experiments, we showed that p18 protein bound to VE-cadherin before Rab11a through its VE-cadherin-binding domain (aa 1–39). Transendothelial resistance showed that overexpression of p18 promoted the circulation of VE-cadherin to adherens junctions and the recovery of the endothelial barrier. Silencing of p18 caused endothelial barrier dysfunction and prevented Rab11a-positive recycling endosome accumulation in the perinuclear recycling compartments. Furthermore, p18 knockdown in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide and cecal ligation puncture. This study showed that p18 regulated the pulmonary endothelial barrier function in vitro and in vivo by regulating the binding of Rab11a to VE-cadherin and the activation of Rab11a.

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Driving Rho GTPase activity in endothelial cells regulates barrier integrity

Thrombosis and Haemostasis ◽

10.1160/th09-06-0403 ◽

2010 ◽

Vol 103 (01) ◽

pp. 40-55 ◽

Cited By ~ 130

Author(s):

Cora Beckers ◽

Victor van Hinsbergh ◽

Geerten van Nieuw Amerongen

Keyword(s):

Barrier Function ◽

Rho Gtpases ◽

Rho Gtpase ◽

Three Dimensional ◽

Regulatory Proteins ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Gtpase Activation ◽

The Individual

SummaryIn the past decade understanding of the role of the Rho GTPases RhoA, Rac1 and Cdc42 has been developed from regulatory proteins that regulate specific actin cytoskeletal structures – stress fibers, lamellipodia and filopodia – to complex integrators of cytoskeletal structures that can exert multiple functions depending on the cellular context. Fundamental to these functions are three-dimensional complexes between the individual Rho GTPases, their specific activators (GEFs) and inhibitors (GDIs and GAPs), which greatly outnumber the Rho GTPases themselves, and additional regulatory proteins. By this complexity of regulation different vasoactive mediators can induce various cytoskeletal structures that enable the endothelial cell (EC) to respond adequately. In this review we have focused on this complexity and the consequences of Rho GTPase regulation for endothelial barrier function. The permeability inducers thrombin and VEGF are presented as examples of G-protein coupled receptor- and tyrosine kinase receptormediated Rho GTPase activation, respectively. These mediators induce complex but markedly different networks of activators, inhibitors and effectors of Rho GTPases, which alter the endothelial barrier function. An interesting feature in this regulation is that Rho GTPases often have both barrier-protecting and barrier-disturbing functions. While Rac1 enforces the endothelial junctions, it becomes part of a barrier-disturbing mechanism as activator of reactive oxygen species generating NADPH oxidase. Similarly RhoA is protective under basal conditions, but becomes involved in barrier dysfunction after activation of ECs by thrombin. The challenge and promise lies in unfolding this complex regulation, as this will provide leads for new therapeutic opportunities.

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Chronic ingestion of alcohol modulates expression of ubiquitin editing enzyme A20 in lung macrophages

Multidisciplinary Respiratory Medicine ◽

10.4081/mrm.2011.457 ◽

2019 ◽

Vol 6 ◽

Author(s):

Quan-Yong Huang ◽

Yu-Chuan Chen ◽

Shui-Ping Liu

Keyword(s):

Evans Blue ◽

Barrier Function ◽

Underlying Mechanism ◽

Endothelial Barrier ◽

Lung Edema ◽

Chronic Alcohol ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Editing Enzyme

Background: Alcohol abuse is involved in the pathogenesis of multiple organ disorders; the underlying mechanism is incompletely understood. The ubiquitin editing enzyme A20 is involved in regulating activities in the cell. Suppression of A20 is suggested as one factor in the initiation of inflammation. This study investigates the mechanism by which chronic alcohol consumption modulates the levels of ubiquitin editing enzyme A20 in macrophages and further contributes to induce endothelial barrier dysfunction in the lung. Methods: Mice were gavage-fed with 40% alcohol daily for 0- 3 weeks. Airway macrophages were collected by lung lavage. Expression of ubiquitin editing enzyme A20 in isolated macrophages was assessed at both mRNA and protein levels. The endothelial barrier function of the lung was evaluated by the Evans blue method. Results: Mice treated with alcohol for 3 weeks showed an increase in cell infiltration in the lung in response to exposure to peptidoglycan; over 80% of the infiltrated cells were macrophages. Furthermore, we observed that A20 level was suppressed in macrophages of mice treated with alcohol; the levels of tumor necrosis factor, interleukin-6 and nuclear factor kappa B in macrophage were increased. In addition, the endothelial barrier function of the lung was compromised, showing excessive infiltration of Evans blue in the lung indicating lung edema. Pretreatment with synthesized A20 inhibited alcohol-induced lung endothelial barrier dysfunction. Conclusions: We conclude that chronic alcohol ingestion disturbs the endothelial barrier function in the lung by modulating macrophage properties. Increase in A20 in the cell may have potential for the treatment of inflammatory disorders.

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