Effects of soybean agglutinin on intestinal barrier permeability and tight junction protein expression in weaned piglets

This study was developed to provide further information on the intestinal barrier permeability and the tight junction protein expression in weaned piglets fed with different levels of soybean agglutinin (SBA). Twenty-five weaned crossbred barrows (Duroc × Landrace × Yorkshire) were selected and randomly allotted to five groups, each group with five replicates. The piglets in the control group were not fed with leguminous products. 0.05, 0.1, 0.15 and 0.2% SBA was added to the control diet to form four experimental diets, respectively. After the experimental period of 7 days (for each group), all the piglets were anesthetized with excess procaine and slaughtered. The d-lactic acid in plasma and the Ileal mucosa diamine oxidase (DAO) was analyzed to observe the change in the intestinal permeability. The tight junction proteins occludin and ZO-1 in the jejunum tissue distribution and relative expression were detected by immunohistochemistry and Western Blot. The results illustrated that a high dose of SBA (0.1-0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no significant affects. The contents of DAO, d-lactic acid, occludin or ZO-1, had a linear relationship with the SBA levels (0-0.2%) in diets. The high dose SBA (0.1-0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no affects.     

An Experimental Workflow for Studying Barrier Integrity,Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.     

β-Conglycinin Reduces the Tight Junction Occludin and ZO-1 Expression in IPEC-J2

Soybean allergy presents a health threat to humans and animals. The mechanism by which food/feed allergen β-conglycinin injures the intestinal barrier has not been well understood. In this study, the changes of epithelial permeability, integrity, metabolic activity, the tight junction (TJ) distribution and expression induced by β-conglycinin were evaluated using IPEC-J2 model. The results showed a significant decrease of trans-epithelial electrical resistance (TEER) (p < 0.001) and metabolic activity (p < 0.001) and a remarkable increase of alkaline phosphatase (AP) activity (p < 0.001) in a dose-dependent manner. The expression levels of tight junction occludin and ZO-1 were decreased (p < 0.05). The reduced fluorescence of targets and change of cellular morphology were recorded. The tight junction occludin and ZO-1 mRNA expression linearly declined with increasing β-conglycinin (p < 0.001).     

Germinated Rhynchosia nulubilis Fermented with Lactobacillus pentosus SC65 Reduces Particulate Matter Induced Type II Alveolar Epithelial Apoptotic Cell Death

Particulate matter (PM) is a significant environmental pollutant that promotes respiratory diseases, including lung injury and inflammation, by inducing oxidative stress. Rhynchosia nulubilis (black soybean) is traditionally used to prevent chronic respiratory disease via inducing antioxidant and anti-inflammatory effects. To investigate the effects of Lactobacillus pentosus SC65 fermented GR (GR-SC65) and Pediococcus pentosaceus ON81A (GR-ON81A) against PM-induced oxidative stress and cell death in A549 cells, we performed the 2-7-dichlorodihydrofluorescein diacetate and cell counting kit-8 assays, as well as Hoechst 33342 and propidium iodide staining and western blotting. GR-SC65 showed the highest total polyphenolic contents and 1,1-diphenyl-2-picrylidrazil radical scavenging activity among lactic acid bacteria-fermented GRs (p < 0.001 vs. GR). Four soy peptides, β-conglycinin breakdowns (INAENNQRNF, ISSEDKPFN, LAFPGSAQAVEK, and LAFPGSAKDIEN), were detected in GR-SC65, but not in GR. In GR-SC65, PM-induced A549 cell death was less than that observed in GR-ON81A and GR (p < 0.001 vs. PM-treated group). GR-SC65 significantly decreased intracellular reactive oxidative species (ROS) when compared with PM (*** p < 0.001 vs. PM). GR-SC65 decreased the levels of BAX, active caspase-9, -3, and poly ADP-ribose polymerase (PARP) proteins (^# p < 0.01, ^### p < 0.001 vs. PM), while increasing the level of BCL-2 protein, a mitochondrial anti-apoptotic protein (^### p < 0.001 vs. PM). Our findings indicate that GR-SC65 inhibited PM-induced cell death by suppressing the levels of ROS, active caspase-9 and -3, and PARP proteins, while enhancing the level of BCL-2 protein in type II alveolar epithelial A549 cells. Therefore, GR-SC65 might be a potential therapeutic and preventive agent against PM-induced lung injury.     

Ischemic Preconditioning Protects against Spinal Cord Ischemia-Reperfusion Injury in Rabbits by Attenuating Blood Spinal Cord Barrier Disruption

Ischemic preconditioning has been reported to protect against spinal cord ischemia-reperfusion (I-R) injury, but the underlying mechanisms are not fully understood. To investigate this, Japanese white rabbits underwent I-R (30 min aortic occlusion followed by reperfusion), ischemic preconditioning (three cycles of 5 min aortic occlusion plus 5 min reperfusion) followed by I-R, or sham surgery. At 4 and 24 h following reperfusion, neurological function was assessed using Tarlov scores, blood spinal cord barrier permeability was measured by Evan’s Blue extravasation, spinal cord edema was evaluated using the wet-dry method, and spinal cord expression of zonula occluden-1 (ZO-1), matrix metalloproteinase-9 (MMP-9), and tumor necrosis factor-α (TNF-α) were measured by Western blot and a real-time polymerase chain reaction. ZO-1 was also assessed using immunofluorescence. Spinal cord I-R injury reduced neurologic scores, and ischemic preconditioning treatment ameliorated this effect. Ischemic preconditioning inhibited I-R-induced increases in blood spinal cord barrier permeability and water content, increased ZO-1 mRNA and protein expression, and reduced MMP-9 and TNF-α mRNA and protein expression. These findings suggest that ischemic preconditioning attenuates the increase in blood spinal cord barrier permeability due to spinal cord I-R injury by preservation of tight junction protein ZO-1 and reducing MMP-9 and TNF-α expression.     

Fingolimod (FTY720-P) Does Not Stabilize the Blood–Brain Barrier under Inflammatory Conditions in an in Vitro Model

Breakdown of the blood-brain barrier (BBB) is an early hallmark of multiple sclerosis (MS), a progressive inflammatory disease of the central nervous system. Cell adhesion in the BBB is modulated by sphingosine-1-phosphate (S1P), a signaling protein, via S1P receptors (S1P₁). Fingolimod phosphate (FTY720-P) a functional S1P₁ antagonist has been shown to improve the relapse rate in relapsing-remitting MS by preventing the egress of lymphocytes from lymph nodes. However, its role in modulating BBB permeability—in particular, on the tight junction proteins occludin, claudin 5 and ZO-1—has not been well elucidated to date. In the present study, FTY720-P did not change the transendothelial electrical resistance in a rat brain microvascular endothelial cell (RBMEC) culture exposed to inflammatory conditions and thus did not decrease endothelial barrier permeability. In contrast, occludin was reduced in RBMEC culture after adding FTY720-P. Additionally, FTY720-P did not alter the amount of endothelial matrix metalloproteinase (MMP)-9 and MMP-2 in RBMEC cultures. Taken together, our observations support the assumption that S1P₁ plays a dual role in vascular permeability, depending on its ligand. Thus, S1P₁ provides a mechanistic basis for FTY720-P-associated disruption of endothelial barriers—such as the blood-retinal barrier—which might result in macular edema.     

Evidence for the Involvement of RhoA Signaling in the Ethanol-Induced Increase in Intestinal Epithelial Barrier Permeability

In this work, we investigated the potential role of the small G protein RhoA in ethanol-induced tight junction (TJ) protein disassembly and increased intestinal epithelial barrier (IEB) permeability. Our study used Caco-2 cells as an in vitro IEB model and RhoA short hairpin RNA (shRNA) interference to establish whether RhoA plays a role in ethanol-induced TJ opening. RhoA shRNA interference partially inhibited epithelial leakage and restored normal transepithelial electrical resistance (TEER) values in the IEB. Moreover, RhoA shRNA interference prevented a shift in occludin distribution from insoluble to soluble fractions. Additionally, RhoA shRNA interference inhibited the ethanol-induced expression of zonula occludens-1 (ZO-1). Finally, RhoA shRNA interference inhibited an ethanol-induced increase in RhoA activity. The contributions of RhoA to an ethanol-induced increase in IEB permeability are associated with TJ disassembly.     

Oxidative Stress Mediates the Disruption of Airway Epithelial Tight Junctions through a TRPM2-PLCγ1-PKCα Signaling Pathway

Oxidative stress has been implicated as an important contributing factor in the pathogenesis of several pulmonary inflammatory diseases. Previous studies have indicated a relationship between oxidative stress and the attenuation of epithelial tight junctions (TJs). In Human Bronchial Epithelial-16 cells (16HBE), we demonstrated the degradation of zonula occludens-1 (ZO-1), and claudin-2 exhibited a great dependence on the activation of the transient receptor potential melastatin (TRPM) 2 channel, phospholipase Cγ1 (PLCγ1) and the protein kinase Cα (PKCα) signaling cascade.     

Nicotinamide Mononucleotide and Coenzyme Q10 Protects Fibroblast Senescence Induced by Particulate Matter Preconditioned Mast Cells

Particulate matter (PM) pollutants impose a certain degree of destruction and toxicity to the skin. Mast cells in the skin dermis could be activated by PMs that diffuse across the blood vessel after being inhaled. Mast cell degranulation in the dermis provides a kind of inflammatory insult to local fibroblasts. In this study, we evaluated human dermal fibroblast responses to conditioned medium from KU812 cells primed with PM. We found that PM promoted the production of proinflammatory cytokines in mast cells and that the cell secretome induced reactive oxygen species and mitochondrial reactive oxygen species production in dermal fibroblasts. Nicotinamide mononucleotide or coenzyme Q10 alleviated the generation of excessive ROS and mitochondrial ROS induced by the conditioned medium from PM-activated KU812 cells. PM-conditioned medium treatment increased the NF-κB expression in dermal fibroblasts, whereas NMN or Q10 inhibited p65 upregulation by PM. The reduced sirtuin 1 (SIRT 1) and nuclear factor erythroid 2-related Factor 2 (Nrf2) expression induced by PM-conditioned medium was reversed by NMN or Q10 in HDFs. Moreover, NMN or Q10 attenuated the expression of senescent β-galactosidase induced by PM-conditioned KU812 cell medium. These findings suggest that NMN or Q10 ameliorates PM-induced inflammation by improving the cellular oxidative status, suppressing proinflammatory NF-κB, and promoting the levels of the antioxidant and anti-inflammatory regulators Nrf2 and SIRT1 in HDFs. The present observations help to understand the factors that affect HDFs in the dermal microenvironment and the therapeutic role of NMN and Q10 as suppressors of skin aging.     

Microglial Activation Damages Dopaminergic Neurons through MMP-2/-9-Mediated Increase of Blood-Brain Barrier Permeability in a Parkinson’s Disease Mouse Model

Chronic neuroinflammation has been considered to be involved in the progressive dopaminergic neurodegeneration in Parkinson’s disease (PD). However, the mechanisms remain unknown. Accumulating evidence indicated a key role of the blood–brain barrier (BBB) dysfunction in neurological disorders. This study is designed to elucidate whether chronic neuroinflammation damages dopaminergic neurons through BBB dysfunction by using a rotenone-induced mouse PD model. Results showed that rotenone dose-dependently induced nigral dopaminergic neurodegeneration, which was associated with increased Evans blue content and fibrinogen accumulation as well as reduced expressions of zonula occludens-1 (ZO-1), claudin-5 and occludin, three tight junction proteins for maintaining BBB permeability, in mice, indicating BBB disruption. Rotenone also induced nigral microglial activation. Depletion of microglia or inhibition of microglial activation by PLX3397 or minocycline, respectively, greatly attenuated BBB dysfunction in rotenone-lesioned mice. Mechanistic inquiry revealed that microglia-mediated activation of matrix metalloproteinases-2 and 9 (MMP-2/-9) contributed to rotenone-induced BBB disruption and dopaminergic neurodegeneration. Rotenone-induced activation of MMP-2/-9 was significantly attenuated by microglial depletion and inactivation. Furthermore, inhibition of MMP-2/-9 by a wide-range inhibitor, SB-3CT, abrogated elevation of BBB permeability and simultaneously increased tight junctions expression. Finally, we found that microglial depletion and inactivation as well as inhibition of MMP-2/-9 significantly ameliorated rotenone-elicited nigrostriatal dopaminergic neurodegeneration and motor dysfunction in mice. Altogether, our findings suggested that microglial MMP-2/-9 activation-mediated BBB dysfunction contributed to dopaminergic neurodegeneration in rotenone-induced mouse PD model, providing a novel view for the mechanisms of Parkinsonism.     

A method for exposing rodents to resuspended particles using whole-body plethysmography

Background  

Epidemiological studies have reported increased risks of cardiopulmonary-related hospitalization and death in association with exposure to elevated levels of particulate matter (PM) across a wide range of urban areas. In response to these findings, researchers have conducted animal inhalation exposures aimed at reproducing the observed toxicologic effects. However, it is technically difficult to quantitate the actual amount of PM delivered to the lung in such studies, and dose is frequently estimated using default respiration parameters. Consequently, the interpretation of PM-induced effects in rodents exposed via whole-body inhalation is often compromised by the inability to determine deposited dose. To address this problem, we have developed an exposure system that merges the generation of dry, aerosolized particles with whole-body plethysmography (WBP), thus permitting inhalation exposures in the unrestrained rat while simultaneously obtaining data on pulmonary function.     

Effect of Quercetin on mitoBKCa Channel and Mitochondrial Function in Human Bronchial Epithelial Cells Exposed to Particulate Matter

Particulate matter (PM) exposure increases reactive oxygen species (ROS) levels. It can lead to inflammatory responses and damage of the mitochondria thus inducing cell death. Recently, it has been shown that potassium channels (mitoK) located in the inner mitochondrial membrane are involved in cytoprotection, and one of the mechanisms involves ROS. To verify the cytoprotective role of mitoBK_Ca, we performed a series of experiments using a patch-clamp, transepithelial electrical resistance assessment (TEER), mitochondrial respiration measurements, fluorescence methods for the ROS level and mitochondrial membrane potential assessment, and cell viability measurements. In the human bronchial epithelial cell model (16HBE14σ), PM < 4 μm in diameter (SRM-PM4.0) was used. We observed that PM decreased TEER of HBE cell monolayers. The effect was partially abolished by quercetin, a mitoBK_Ca opener. Consequently, quercetin decreased the mitochondrial membrane potential and increased mitochondrial respiration. The reduction of PM-induced ROS level occurs both on cellular and mitochondrial level. Additionally, quercetin restores HBE cell viability after PM administration. The incubation of cells with PM substantially reduced the mitochondrial function. Isorhamnetin had no effect on TEER, the mitoBK_Ca activity, respiratory rate, or mitochondrial membrane potential. Obtained results indicate that PM has an adverse effect on HBE cells at the cellular and mitochondrial level. Quercetin is able to limit the deleterious effect of PM on barrier function of airway epithelial cells. We show that the effect in HBE cells involves mitoBK_Ca channel-activation. However, quercetin’s mechanism of action is not exclusively determined by modulation of the channel activity.     

NMR-Guided Repositioning of Non-Steroidal Anti-Inflammatory Drugs into Tight Junction Modulators

Bioavailability is a major bottleneck in the clinical application of medium molecular weight therapeutics, including protein and peptide drugs. Paracellular transport of these molecules is hampered by intercellular tight junction (TJ) complexes. Therefore, safe chemical regulators for TJ loosening are desired. Here, we showed a potential application of select non-steroidal anti-inflammatory drugs (NSAIDs) as TJ modulators. Based on our previous observation that diclofenac and flufenamic acid directly bound various PDZ domains with a broad specificity, we applied solution nuclear magnetic resonance techniques to examine the interaction of other NSAIDs and the first PDZ domain (PDZ1) of zonula occludens (ZO)-1, ZO-1(PDZ1). Inhibition of ZO-1(PDZ1) is expected to provide loosening of the epithelial barrier function because the domain plays a crucial role in maintaining TJ integrity. Accordingly, diclofenac and indomethacin were found to decrease the subcellular localization of claudin (CLD)-2 but not occludin and ZO-1 at the apicolateral intercellular compartment of Madin–Darby canine kidney (MDCK) II cells. These NSAIDs exhibited 125–155% improved paracellular efflux of fluorescein isothiocyanate insulin for the Caco-2 cell monolayer. We propose that these NSAIDs can be repurposed as drug absorption enhancers for peptide drugs.     

Alterations in cell cholesterol content modulate Ca2+-induced tight junction assembly by MDCK cells

Transepithelial electrical resistance (TER), a measure of tight junction (TJ) barrier function, develops more rapidly and reaches higher values after preincubation of MDCK cells for 24 h with 2 μM Lovastatin (lova), an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase. While this effect was attributed to a 30% fall in cholesterol (CH), possible effects of lova on the supply of prenyl group precursors could not be excluded. In the current study, strategies were devised to examine effects on TER of agents that simultaneously lower CH and increase the flux of intermediates through the CH biosynthetic pathway. Zaragozic acid, 20 μM, an inhibitor of squalene synthase known to increase the synthesis of isoprenoids and levels of prenylated proteins, lowered cell CH by 30% after 24 h, while accelerating development of TER in the same manner as lova. TER was also enhanced, despite a 23% increase in the rate of [³H]acetate incorporation into CH, when total CH was reduced by 45% during a 2-h incubation with 2 mM methyl β-cyclodextrin (MBCD), an agent that stimulates CH efflux from cells. The fact that the rate of TER development was diminished when cell CH content was elevated by incubation with a complex of CH and MBCD is further evidence that this sterol modulates development of the epithelial barrier. Cell associated CH derived from the complex was similar to endogenous CH with respect to its accessibility to cholesterol oxidase. Lova's effect on TER was diminished when 5 μg/mL of CH was added to the medium during the last 11 h of incubation with lova.     

Midazolam Ameliorates Hyperglycemia-Induced Glomerular Endothelial Dysfunction by Inhibiting Transglutaminase 2 in Diabetes

Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca²⁺ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2^−/− mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.     

Real-Time Monitoring the Effect of Cytopathic Hypoxia on Retinal Pigment Epithelial Barrier Functionality Using Electric Cell-Substrate Impedance Sensing (ECIS) Biosensor Technology

Disruption of retinal pigment epithelial (RPE barrier integrity is a hallmark feature of various retinal blinding diseases, including diabetic macular edema and age-related macular degeneration, but the underlying causes and pathophysiology are not completely well-defined. One of the most conserved phenomena in biology is the progressive decline in mitochondrial function with aging leading to cytopathic hypoxia, where cells are unable to use oxygen for energy production. Therefore, this study aimed to thoroughly investigate the role of cytopathic hypoxia in compromising the barrier functionality of RPE cells. We used Electric Cell-Substrate Impedance Sensing (ECIS) system to monitor precisely in real time the barrier integrity of RPE cell line (ARPE-19) after treatment with various concentrations of cytopathic hypoxia-inducing agent, Cobalt(II) chloride (CoCl₂). We further investigated how the resistance across ARPE-19 cells changes across three separate parameters: R_b (the electrical resistance between ARPE-19 cells), α (the resistance between the ARPE-19 and its substrate), and C_m (the capacitance of the ARPE-19 cell membrane). The viability of the ARPE-19 cells and mitochondrial bioenergetics were quantified with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and seahorse technology, respectively. ECIS measurement showed that CoCl₂ reduced the total impedance of ARPE-19 cells in a dose dependent manner across all tested frequencies. Specifically, the ECIS program’s modelling demonstrated that CoCl₂ affected R_b as it begins to drastically decrease earlier than α or C_m, although ARPE-19 cells’ viability was not compromised. Using seahorse technology, all three concentrations of CoCl₂ significantly impaired basal, maximal, and ATP-linked respirations of ARPE-19 cells but did not affect proton leak and non-mitochondrial bioenergetic. Concordantly, the expression of a major paracellular tight junction protein (ZO-1) was reduced significantly with CoCl_2-treatment in a dose-dependent manner. Our data demonstrate that the ARPE-19 cells have distinct dielectric properties in response to cytopathic hypoxia in which disruption of barrier integrity between ARPE-19 cells precedes any changes in cells’ viability, cell-substrate contacts, and cell membrane permeability. Such differences can be used in screening of selective agents that improve the assembly of RPE tight junction without compromising other RPE barrier parameters.     

Zearalenone-Induced Mechanical Damage of Intestinal Barrier via the RhoA/ROCK Signaling Pathway in IPEC-J2 Cells

Zearalenone (ZEN) is a widespread contaminant of cereals and agricultural products which causes food safety issues. Ingesting food or feed contaminated with ZEN can disrupt the intestinal epithelial barrier function. The RhoA/ROCK signaling pathway plays a key role in regulating the epithelial barrier function, but studies on such roles have rarely focused on the intestine. The aim of this experiment was to investigate the exact mechanism of ZEN-induced intestinal barrier damage and whether the RhoA/ROCK signaling pathway is involved. The results showed that ZEN significantly induced alkaline phosphatase (AP) activity and FITC–dextran (4 kDa) passage across the epithelial barrier, which significantly reduced the transepithelial resistance (TEER). Meanwhile, ZEN could induce the significantly down-regulated mRNA expression of tight junction proteins (occludin, claudin-1, ZO-1, and claudin-3) and redistribution of ZO-1 immunofluorescence. Further studies demonstrated that ZEN exposure activated the RhoA/ROCK signaling pathway, significantly up-regulated the mRNA expression of ROCK1, the main effector of the signaling pathway, the protein expression of phosphorylated myosin light chain (MLC) and myosin light chain kinase (MLCK), and relatively increased the activity of ATP in cells, simultaneously remodeling the cytoskeleton (F-actin). Overall, our study indicated that ZEN induced intestinal barrier dysfunction by activating the RhoA/ROCK signaling pathway.     

Oxidized Lipoprotein(a) Increases Endothelial Cell Monolayer Permeability via ROS Generation

Oxidized lipoprotein(a) (oxLp(a)) is a more potent marker of atherogenesis than native Lp(a). However, the molecular mechanisms of oxLp(a) activity are not clear. Reactive oxygen species (ROS) have recently been suggested as acting as intracellular second messengers. In this study, the effects of oxLp(a) on endothelial cell monolayer permeability and the role of reactive oxygen species (ROS) generation in these effects were investigated. Our results showed that oxLp(a) inhibited desmoglein-1 (DSG1) and desmocollin-2 (DSC2) expression at both mRNA and protein levels in a dose- and time-dependent manner, and increased the generation of cellular ROS. Down-regulation of DSG1 and DSC2 was strengthened by pretreatment with H₂O₂ and attenuated by superoxide dismutase (SOD) treatment. Furthermore, oxLp(a) increased endothelial cell monolayer permeability, and this effect was enhanced by H₂O₂ and blunted by SOD. Taken together, these results demonstrate that oxLp(a) increases endothelial cell monolayer permeability, which is mediated at least in part via ROS generation.