Cell Tight Junctions Research Articles

Targeting Cancer Cell Tight Junctions Enhances PLGA-Based Photothermal Sensitizers' Performance In Vitro and In Vivo.

The development of non-invasive photothermal therapy (PTT) methods utilizing nanoparticles as sensitizers is one of the most promising directions in modern oncology. Nanoparticles loaded with photothermal dyes are capable of delivering a sufficient amount of a therapeutic substance and releasing it with the desired kinetics in vivo. However, the effectiveness of oncotherapy methods, including PTT, is often limited due to poor penetration of sensitizers into the tumor, especially into solid tumors of epithelial origin characterized by tight cellular junctions. In this work, we synthesized 200 nm nanoparticles from the biocompatible copolymer of lactic and glycolic acid, PLGA, loaded with magnesium phthalocyanine, PLGA/Pht-Mg. The PLGA/Pht-Mg particles under the irradiation with NIR light (808 nm), heat the surrounding solution by 40 °C. The effectiveness of using such particles for cancer cells elimination was demonstrated in 2D culture in vitro and in our original 3D model with multicellular spheroids possessing tight cell contacts. It was shown that the mean inhibitory concentration of such nanoparticles upon light irradiation for 15 min worsens by more than an order of magnitude: IC50 increases from 3 µg/mL for 2D culture vs. 117 µg/mL for 3D culture. However, when using the JO-4 intercellular junction opener protein, which causes a short epithelial–mesenchymal transition and transiently opens intercellular junctions in epithelial cells, the efficiency of nanoparticles in 3D culture was comparable or even outperforming that for 2D (IC50 = 1.9 µg/mL with JO-4). Synergy in the co-administration of PTT nanosensitizers and JO-4 protein was found to retain in vivo using orthotopic tumors of BALB/c mice: we demonstrated that the efficiency in the delivery of such nanoparticles to the tumor is 2.5 times increased when PLGA/Pht-Mg nanoparticles are administered together with JO-4. Thus the targeting the tumor cell junctions can significantly increase the performance of PTT nanosensitizers.

Renal-on-Chip Microfluidic Platform with a Force-Sensitive Resistor (ROC-FS) for Molecular Pathogenesis Analysis of Hydronephrosis.

Hydronephrosis is one of the most common diseases in urology. However, due to the difficulties in clinical trials and the lack of reliable in vitro platforms, the surgical indicators are not clear. Herein, the renal-on-chip with a force-sensitive resistor microfluidic platform was established to simulate the state of hydronephrosis. Cell counting kit-8 (CCK-8) and tight junction protein claudin-2 were detected on a renal-on-chip microfluidic platform with a force-sensitive resistor (ROC-FS). The results indicated that the ROC-FS had normal physiological functions and the cell viability on ROC-FS declined to around 40% after 48 h of hydronephrosis-simulated treatment. In addition, proteomics analysis of 15 clinical ureteropelvic junction obstruction (UPJO) samples showed that compared with normal children, a total of 50 common proteins were differentially expressed in UPJO children (P < 0.05, |log2fold change| ≥ 1). Metabolomic analysis of 39 clinical UPJO samples showed that a total of 241 metabolisms were dysregulated. Subsequent immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analysis using ROC-FS were performed to identify the clinical multi-omics results for screening. All results pointed out that the TGF-β-related signaling pathways and arginine-related metabolism signaling pathways were dysregulated and α-SMA, AGT, and AGA might be the potential biomarkers of hydronephrosis. In addition, correlation analysis of AGT and KLK1 with differential renal function (DRF) from clinical samples indicated good correlation coefficients (R2 0.923, 0.8742, 0.6412, and 0.8347). This demonstrates the state of hydronephrosis could be significantly correlated with the biomarkers. These findings could provide a reliable reference for determining surgical biomarkers clinically, and ROC could be further used in the analysis of other kidney diseases.

Membrane proteomic analysis identifies the polarity protein PARD3 as a novel antiviral protein against PEDV infection

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. PEDV could remodel host membrane structures for their replication, assembly and escape from host cells. However, little is known about the host membrane proteins of PEDV infection. In this study, we analyzed differentially abundant proteins (DAPs) between PEDV infection group and control group and identified the polarity protein PARD3 as one of the most significantly DAPs. PARD3 is implicated in the formation of tight junctions at epithelial cell-cell contacts. Then, we found that PEDV infection promoted the degradation of PARD3 via the ubiquitin proteasome pathway. Moreover, knockdown of PARD3 promoted the proliferation of PEDV. Further study showed that the downregulation of PARD3 altered the normal morphology of the tight junction proteins and promoted apical and basolateral virus proliferation. Tight junctions enable epithelial cells to form physical barriers, which act as an innate immune mechanism that can impede viral infection and PEDV affected the barrier functions by causing degradation of PARD3. Taken together, this work is the first time to investigate the membrane protein profile of PEDV-infected cells using quantitative proteomics and suggests that PARD3 could be a potential novel antiviral protein against PEDV infection. SignificanceMembrane proteins are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are changed during in response to environmental stress. However, membrane proteins are difficult to study because of their hydrophobicity. Membrane proteomic methods using mass spectrometry analysis have been developed and applied for the characterization of the plasma membrane and subcellular organelles of various virus infected cells. Porcine epidemic diarrhea virus (PEDV) is an enteric pathogen of importance to the swine industry, causing high mortality in neonatal piglets. Because PEDV infected Vero cells can lead to significant changes in cell membrane morphology and form syncytial lesions. Here, we isolated the membrane proteins of PEDV infected and control cells and applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantitatively identify the differentially abundant proteins (DAPs) in PEDV-infected Vero cells and confirmed the DAPs by performing RT-qPCR and Western blot analysis. Among these differential proteins, we focused on a down-regulated protein PARD3 which is important for cell tight junction and cell polarity. Loss of PARD3 can destroy the tight junction of cells and promote the proliferation of PEDV in the apical and basolateral sides. These findings will provide valuable information to better understand the mechanisms underlying the host defense responses to PEDV infection.

LncRNA FGD5-AS1 accelerates intracerebral hemorrhage injury in mice by adsorbing miR-6838-5p to target VEGFA

Intracerebral hemorrhage (ICH) can usually cause severe neuroinflammation and blood-brain barrier (BBB) damage. Previous studies supported the important role of long non-coding RNAs (lncRNAs) in ICH treatment. This study aimed to explore the effect of lncRNA FGD5 antisense RNA 1 (FGD5-AS1) on ICH and its potential molecular mechanisms. C57BL/6 mice were injected with collagenase VII to establish an ICH mice model. In addition, brain cerebral microvascular endothelial cells (BMVECs) were treated by oxygen-glucose deprivation (OGD)/hemin to simulate ICH. RT-qPCR revealed that FGD5-AS1 was upregulated in serum of ICH patients and mice and in OGD/hemin-treated BMVECs. Luciferase reporter gene and pull-down assays predicted and verified that FGD5-AS1 bound to miR-6838-5p, and VEGFA was a target of miR-6838-5p. FGD5-AS1 knockdown decreased the inflammatory factor contents in brain tissues and BMVECs. FGD5-AS1 overexpression inhibited cell proliferation, invasion and tight junction protein levels, and promoted apoptosis, increased the permeability of BBB and secretion of pro-inflammatory factors. In addition, miR-6838-5p knockdown reversed the inhibitory effect of FGD5-AS1 knockdown on the PI3K/Akt signaling pathway. In conclusion, FGD5-AS1 may act as an important regulator to promote apoptosis, cell permeability and inflammatory response of BMVECs via the miR-6838-5p/VEGFA axis in ICH mice.

DL-methionine and DL-methionyl-DL-methionine increase intestinal development and activate Wnt/β-catenin signaling activity in domestic pigeons (Columba livia)

This experiment was undertaken to investigate the effects of parental dietary DL-methionine (DL-Met) and DL-methionyl-DL-methionine (DL-Met-Met) supplementation on the intestinal development of young squabs. A total of 108 pairs of breeding pigeons and 432 one-day-old squabs were randomly divided into 3 groups: the control group (CON) was fed a basal diet (CP = 15%) and the experimental groups were fed a basal diet supplemented with 0.3% DL-Met or DL-Met-Met. Each pair of breeding pigeons nourished 4 young squabs, and 8 squabs from each treatment were randomly sampled at the end of the experiment. The results indicated that DL-Met and DL-Met-Met supplementation improved the intestinal morphology and structure in the squabs, as reflected by the increased relative intestinal weight of each small intestinal segment, villus height, and villus to crypt ratio. In addition, DL-Met and DL-Met-Met supplementation significantly increased the protein expression of cell proliferation markers (Ki67 and PCNA) and tight junction proteins (ZO-1 and Claudin-1) in the jejunum and strengthened the fluorescence signal intensity of Ki67, PCNA and Villin. Moreover, the expression of Wnt/β-catenin signaling pathway-related proteins (Frizzled 7 [FZD7], p-GSK-3β, Active β-catenin, β-catenin, TCF4, c-Myc, and Cyclin D1), and intestinal peptide transporter 1 (PepT1) in the jejunum was considerably higher in the treatment group than in the CON group (P < 0.05), with the DL-Met-Met group having the highest expression. Consistently, the molecular docking results predicted the possibility that DL-Met or DL-Met-Met binds to the membrane receptor FZD7, which mediates Wnt/β-catenin signaling. Collectively, the improvement of the intestinal development in squabs after parental dietary 0.3% DL-Met and DL-Met-Met supplementation could be through activation of Wnt/β-catenin signaling pathway, and DL-Met-Met is superior to DL-Met. Our findings may provide basic data for further optimizing the feeding formula of breeding pigeons and improving the growth and development of squabs.

Layered Double Hydroxide Modified with Deoxycholic and Hyaluronic Acids for Efficient Oral Insulin Absorption.

IntroductionThis study aimed to construct a layered double hydroxide (LDH) nanoparticle delivery system that was modified by deoxycholic acid (DCA) and hyaluronic acid (HA) to increase the bioavailability of oral insulin.MethodsLDH-DCA-HA was synthesized by the hybridization of DCA and HA with LDH. Subsequently, insulin was loaded onto LDH-DCA-HA, resulting in the formation of INS@LDH-DCA-HA. The in vivo and in vitro mechanisms of insulin release, as well as the efficiency of insulin absorption, were analyzed before and after DCA-HA modification.ResultsMTT assay showed that there was satisfactory biocompatibility between LDH-DCA-HA and Caco-2 cells at a concentration below 1000 μg/mL. Flow cytometry analysis revealed that Caco-2 cells absorbed INS@LDH-DCA-HA more readily than insulin. Measurement of transepithelial electrical resistance indicated that INS@LDH-DCA-HA induced the reversible opening of tight cell junctions, thereby facilitating its absorption. This was confirmed via laser confocal microscopy analysis, revealing that a large amount of zonula occludens-1 tight junction (TJ) protein was utilized for the paracellular pathway of nanoparticles. We also measured the blood glucose levels of type I diabetic mice and found that oral INS@LDH-DCA-HA exerted a steady hypoglycemic effect lasting 12 h, with a small range of postprandial blood glucose fluctuation. Immunofluorescence analysis showed that the strong penetration ability of INS@LDH-DCA-HA allowed insulin to enter epithelial cells more readily than free insulin. Finally, immunohistochemical analysis of anti-SLC10A1 protein confirmed that the cholic acid transporter receptor protein played a key role in the functioning of INS@LDH-DCA-HA.ConclusionLDH nanoparticles modified by DCA and HA improved the absorption efficiency of insulin by opening the TJs of cells and interacting with the cholic acid transporter receptor protein.

The ameliorative effect of melatonin on LPS-induced Sertoli cells inflammatory and tight junctions damage via suppression of the TLR4/MyD88/NF-κB signaling pathway in newborn calf

The blood-testicular barrier (BTB) is involved in spermatogenesis, protects sperm development, and plays a crucial role in the reproductive process. Tight junctions (TJs) between Sertoli cells (SCs) are the key structure of (BTB), and if its structure is damaged, BTB function is affected. The cellular inflammation caused by Gram-negative bacteria affects the structural integrity of TJs. Melatonin (MT) has anti-inflammatory effects; however, the effect of MT in newborn calf SCs is unknown. Therefore, this experiment studied the protective effect of MT. The results showed that LPS upregulated TLR4, MyD88, and NF-κB expressions, in turn, activated the TLR4/MyD88/NF-κB signaling pathway, produced a large amount of IL-6 and IL-1β, downregulated the expression of ZO-1 and Occludin, and reduced the viability of SCs, which resulted in the inflammatory response of SCs and damage of TJs. The addition of MT decreased TLR4, MyD88, and NF-κB expressions, it then inhibited the activation of TLR4/MyD88/NF-κB signaling pathway, downregulated the expression of IL-6 and IL-1β, upregulated the expression of ZO-1 and Occludin, and increased the cell viability, thereby alleviating the inflammatory response of SCs, and restored the TJs structure. Overall, our results reveal that MT can alleviate LPS-induced in newborn calf SCs Inflammation and TJs injury through TLR4/MyD88/NF-κB signaling pathway.

IL-1β is a key inflammatory cytokine that weakens lactation-specific tight junctions of mammary epithelial cells

In lactating mammary glands, alveolar mammary epithelial cells (MECs) produce milk and form less-permeable tight junctions (TJs). However, alveolar TJs are weakened with a reduction in milk production in mammary glands due to mastitis or weaning in the presence of high levels of IL-1β, IL-6, or TNF-α. In this study, using in vitro cultured model of MECs with milk-producing ability and lactation-specific TJs, we investigated whether the aforementioned cytokines affect MEC TJs. The results showed that TNF-α, IL-1β, and IL-6 affected lactation-specific TJs in different ways. In particular, upon activation of p38 and JNK signalling, IL-1β caused rapid disruption of TJs at tricellular contact points. IL-1β treatment led to decreased CLDN3, CLDN4, and OCLN levels and a weakened TJ barrier. The adverse effects of IL-1β on TJs were mimicked by anisomycin, which is an activator of p38 and JNK signalling, and were blocked by MEC pretreatment with a p38 inhibitor but not a JNK inhibitor. The mislocalization of tricellulin at tricellular contact areas was confirmed in MECs treated with IL-1β or anisomycin. These results indicate that IL-1β is a key cytokine that adversely affects the TJs between MECs by activating p38.

Claudin-9 constitutes tight junctions of folliculo-stellate cells in the anterior pituitary gland

The anterior pituitary gland regulates growth, metabolism, and reproduction by secreting hormones. Folliculo-stellate (FS) cells are non-endocrine cells located among hormone-producing cells in the anterior pituitary glands. They form follicular lumens, which are sealed by tight junctions (TJs). Although FS cells are hypothesized to contribute to fine-tuning of endocrine cells, little is known about the exact roles of FS cells. Here, we investigated the molecular composition of TJs in FS cells. We demonstrated that occludin is a good marker for TJs in the pituitary gland and examined the structure of the lumens surrounded by FS cells. We also found that claudin-9 is a major component of TJs in the FS cells. In immunoelectron microscopy, claudin-9 was specifically localized at TJs of the FS cells. The expression of claudin-9 was gradually increased in the pituitary gland after birth, suggesting that claudin-9 is developmentally regulated and performs some specific functions on the paracellular barrier of follicles in the pituitary gland. Furthermore, we found that angulin-1, angulin-2, and tricellulin are localized at the tricellular contacts of the FS cells. Our findings provide a first comprehensive molecular profile of TJs in the FS cells, and may lead us towards unveiling the FS cell functions.

Mucoadhesive phenylboronic acid conjugated chitosan oligosaccharide-vitamin E copolymer for topical ocular delivery of voriconazole: Synthesis, in vitro/vivo evaluation, and mechanism

Topical eye drops still face challenges of low-drug treatment effects and frequent dosing in ophthalmic applications due to the low preocular retention rate and low transcorneal permeability. Thus, we designed and synthesized a phenylboronic acid conjugated chitosan oligosaccharide-vitamin E copolymer (PBA-CS-VE) for use in mucoadhesive voriconazole (VRC)-loaded nanomicelles for fungal keratitis. In vitro mucin binding and ex vivo eyeball adhesion tests show that the copolymer has strong mucoadhesion. The transportation of coumarin-6 (C6) across a monolayer of HCE-T cells and 3D cell spheroids confirm the strong corneal penetration ability of PBA-CS-VE. The mechanism of promoting corneal penetration was studied in terms of intracellular calcium-ion concentration, cell membrane potential, cell membrane fluidity, and the tight junctions of cells. The pharmacokinetics in the aqueous humor were examined to evaluate the ability of nanomicelles in promoting corneal penetration and prolonging ocular retention. VRC-loaded PBA-CS-VE nanomicelles (PBA-CS-VE-VRC) yielded a very favorable therapeutic effect on a rabbit model of fungal keratitis in vivo as compared to the free drug. Overall, the results indicate that PBA-CS-VE nanomicelles are a mucoadhesive candidate with enhanced transcorneal permeability and prolonged preocular retention for efficient delivery of topical ocular drugs. Statement of significanceAlthough eye drops are widely used in ocular drug delivery, the disadvantages such as short retention time and weak corneal penetrating ability still seriously affect the therapeutic effect of the drug. Therefore, the mucoadhesive carrier seems to be an interesting strategy for ocular drug delivery. Herein, a novel phenylboronic acid conjugated chitosan oligosaccharide-vitamin E copolymer was designed and constructed as mucoadhesive nanomicelles loaded with voriconazole for fungal keratitis. These nanomicelles were able to improve the in vitro mucin binding and to prolong the residence time of the drug on the surface of the eyeball. Moreover, the nanomicelles exhibited an enhanced drug permeability in cell monolayer models and 3D cell culture models. This work provides a promising ocular drug delivery system.

Phillyrin Prevents Neuroinflammation-Induced Blood-Brain Barrier Damage Following Traumatic Brain Injury via Altering Microglial Polarization.

Background: Phillyrin (Phi) is the main polyphenolic compound found in Forsythia suspensa. Recent studies have revealed that Phi has potent antioxidative and anti-inflammatory effects. However, whether Phi could relieve blood–brain barrier (BBB) damage following traumatic brain injury (TBI) remains unknown. Materials and Methods: Lipopolysaccharide (LPS) was used to activate primary microglia, which were then treated with different doses of Phi or the peroxisome proliferator–activated receptor-gamma (PPARγ) antagonist (GW9662). CCK-8 assay was used for evaluating cell viability, and the cytokines (including IL-1β, IL-6, TNFα, IL-4, IL-10, and TGFβ), microglial phenotypic markers (iNOS, COX2, and CD86 for “M1” polarization; Arg1, Ym1, and CD206 for “M2” polarization), PPARγ, and NF-κB were determined by RT-PCR, Western blot, or cellular immunofluorescence. Primary cultured mouse brain microvascular endothelial cells (BMECs) were stimulated by the condition medium (CM) from microglia. The cell viability, angiogenesis, and tight junction of BMECs were determined via CCK-8 assay, tube formation assay, and Western blot (for detecting MMP3, MMP9, ZO1, claudin-5, and occludin). Furthermore, the mouse TBI model was constructed and treated with Phi and/or GW9662. The BBB integrity was evaluated by H&E staining, Evans blue staining, and tissue immunofluorescence. Results: Phi markedly restrained the pro-inflammatory (“M1” state) cytokines and promoted anti-inflammatory (“M2” polarization) cytokines in LPS-mediated microglia. Phi mitigated “M1” polarization and promoted “M2” polarization of microglia via enhancing PPARγ and inhibiting the NF-κB pathway. The PPARγ antagonist GW9662 significantly repressed Phi-mediated anti-inflammatory effects. Meanwhile, Phi enhanced the viability, tube formation ability, and cell junction of BMECs. In the TBI mouse model, Phi promoted “M2” polarization, whereas it repressed the “M1” polarization of microglia. In addition, Phi reduced TBI-mediated BBB damage. However, the protective effects of Phi were reversed mainly by GW9662 treatment. Conclusion: Phi prevents BBB damage via inhibiting the neuroinflammation of microglia through the PPARγ/NF-κB pathway, which provides a potential therapeutic drug against TBI.

Down-Regulated CLDN10 Predicts Favorable Prognosis and Correlates With Immune Infiltration in Gastric Cancer.

Background: CLDN10, an important component of the tight junctions of epithelial cells, plays a crucial role in a variety of tumors. The effect of CLDN10 expression in gastric cancer, however, has yet to be elucidated. Methods: Differential expression of CLDN10 at the mRNA and protein levels was evaluated using Oncomine, ULCAN, HPA and TIMER2.0 databases. Real-time polymerase chain reaction (RT-PCR) was utilized to further verify the expression of CLDN10 in vitro. Correlations between CLDN10 expression and clinical outcomes of gastric cancer were explored by Kaplan-Meier Plotter. Gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) were performed via LinkedOmics and GeneMANIA. The correlations between CLDN10 expression and immune cell infiltration and somatic copy number alternations (SCNA) in gastric cancer were explored by TIMER2.0 and GEPIA2.0. Results: CLDN10 expression was lower in gastric cancer compared to adjacent normal tissues, and associated with better prognosis. CLDN10 also showed significant differences at different T stages, Lauren classification, treatments and HER2 status. PPI and GSEA analysis showed that CLDN10 might be involved in signal transmission, transmembrane transport and metabolism. In some major immune cells, low expression of CLDN10 was associated with increased levels of immune cell infiltration. In addition, it was found that different SCNA status in CLDN10 might affect the level of immune cell infiltration. Furthermore, the expression of CLDN10 was significantly associated with the expression of several immune cell markers, especially B cell markers, follicular helper T cell (Tfh) markers and T cell exhaustion markers. Conclusion: Down-regulated CLDN10 was associated with better overall survival (OS) in gastric cancer. And CLDN10 may serve as a potential prognostic biomarker and correlate to immune infiltration levels in gastric cancer.

IGF-1 alleviates CCL4-induced hepatic cirrhosis and dysfunction of intestinal barrier through inhibition TLR4/NF-κB signaling mediated by down-regulation HMGB1

Introduction and objectivesCirrhosis has gradually become a serious public health issue, especially the national prevalence of cirrhosis was 29.2% in northwest China. Recent evidence has revealed that intestinal barrier (IB) dysfunction results from and contributes to cirrhosis. Our previous results have indicated that insulin-like growth factors (IGF-1) improved the impaired IB function and downregulated high mobility group protein box-1 (HMGB-1). Nevertheless, the role of the IGF-1/HMGB1 axis in cirrhosis remains largely unknown. Materials and methodsWestern blotting and qRT-PCR were used to detect protein and mRNA levels of related genes. The levels of AST, ALT, IL-1β, and TNF-α were examined using commercial kits. Immunofluorescence was used to evaluate the expression of HMGB1 in tissues. ResultsIn carbon tetrachloride (CCl4)-treated rat, the levels of AST (380.12 vs. 183.97), ALT (148.12 vs. 53.56), IL-1β (155.94 vs. 55.60), and TNF-α (155.00 vs. 48.90) were significantly increased compared with the control group, while IGF-1 treatment significantly alleviated CCL4-induced inflammatory response and IB dysfunction by downregulating HMGB1-mediated the TLR4/MyD88/NF-κB signaling pathway. In vitro experiments, HMGB1 treatment promoted inflammatory cytokines secretion and reduced cell viability and tight junctions by activating the TLR4/MyD88/NF-κB signaling pathway in Caco-2 cells, but IGF-1 alleviated these effects. ConclusionOur findings suggest that IGF-1 might serve as a potential therapeutic target for cirrhosis and IB dysfunction via inactivation of the TLR4/MyD88/NF-κB pathway through down-regulation HMGB1.

Trimellitic anhydride facilitates transepithelial permeability disrupting tight junctions in sinonasal epithelial cells

Trimellitic anhydride (TMA) is a chemical agent classified as a low molecular weight (LMW) agent causing occupational rhinitis (OR) or asthma. Although TMA is recognized as a respiratory sensitizer, the direct and non-immunologic effects of TMA remain unclear. Air- liquid interface (ALI) cultured human nasal epithelial cells (HNECs) derived from control subjects were treated with TMA, followed by measurement of the transepithelial electrical resistance (TEER), paracellular permeability of fluorescein isothiocyanate (FITC)-dextran and immunofluorescence of tight junction proteins claudin-1 and zonula occludens-1 (ZO-1). The cytotoxicity of TMA was evaluated by lactate dehydrogenase (LDH) assay. TMA at concentrations of 2 and 4 mg/mL significantly reduced the TEER within 10 min (p = 0.0177 on 2 mg/mL; p < 0.0001 on 4 mg/mL). The paracellular permeability of FITC-dextran was significantly increased upon challenge with 4 mg/mL TMA for 3 h (p = 0.0088) and 6 h (p = 0.0004). TMA treatment induced a reduction in the fluorescence intensity of claudin-1 and ZO-1 in a dose-dependent manner. LDH assay revealed 4 mg/mL TMA induced cytotoxicity only after 6 h incubation, while 1 or 2 mg/mL TMA caused no cytotoxicity. Our results suggest that TMA has a potential to penetrate the epithelial barrier by disrupting claudin-1 and ZO-1, indicating an important role for sensitization and OR development.

Towards Cellular Ultrastructural Characterization in Organ-on-a-Chip by Transmission Electron Microscopy

Organ-on-a-chip technology is a 3D cell culture breakthrough of the last decade. This rapidly developing field of bioengineering intertwined with microfluidics provides new insights into disease development and preclinical drug screening. So far, optical and fluorescence microscopy are the most widely used methods to monitor and extract information from these models. Meanwhile transmission electron microscopy (TEM), despite its wide use for the characterization of nanomaterials and biological samples, remains unexplored in this area. In our work we propose a TEM sample preparation method, that allows to process a microfluidic chip without its prior deconstruction, into TEM-compatible specimens. We demonstrated preparation of tumor blood vessel-on-a-chip model and consecutive steps to preserve the endothelial cells lining microfluidic channel, for the chip’s further transformation into ultrathin sections. This approach allowed us to obtain cross-sections of the microchannel with cells cultured inside, and to observe cell adaptation to the channel geometry, as well as the characteristic for endothelial cells tight junctions. The proposed sample preparation method facilitates the electron microscopy ultrastructural characterization of biological samples cultured in organ-on-a-chip device.

Study on the Mechanism of Capillary Leakage Caused by Hypoxia-Inducible Factor-1α through Inducing High Expression of Matrix Metalloproteinase-9.

Purposes This study mainly explored the mechanism of capillary leakage caused by hypoxia-inducible factor-1α through inducing high expression of matrix metalloproteinase-9. Method. We established a monolayer endothelial cell model by culturing human umbilical vein endothelial cells (HUVEC) in vitro, used tumor necrosis factor (TNFα) and HIF-1α inhibitor 2-methoxyestradiol (2ME2) to act on HUVEC, and at the same time constructed siRNA-transfected HUVEC to interfere with the expression of HIF-1α. The permeability of monolayer endothelial cells was measured by transwell chamber method, the concentration of MMP-9 in the supernatant was measured by ELISA method, the expression of key molecules related to permeability (HIF- 1α, MMP-9, claudin-5, and ZO-1) was measured by RT-PCR and Western blot method, and the localization and expression of claudin-5 and ZO-1 were measured by immunofluorescence method. We searched for 7 HIF-1α hypoxia response elements within 4000 bp before the transcription start site in the MMP-9 promoter region, constructed the MMP-9 promoter-luciferase reporter gene recombinant plasmid, transfected and stimulated HUVEC with TNFα, and detected the effect of 7 hypoxia response element plasmids on the transcription activity of MMP-9 promoter. Results Under the action of TNFα, the permeability of monolayer endothelial cells increased, and the concentration of MMP-9 in the cell supernatant increased. 2ME2 and HIF-1α-siRNA transfection can improve the above situation (P < 0.05). 2ME2 and HIF-1α-siRNA transfection can inhibit the high expression of HIF-1α and MMP-9 caused by TNFα, thereby increasing the expression of claudin-5 and ZO-1 (P < 0.05). 2ME2 and HIF-1α-siRNA transfection can reduce the inhibition of TNFα on the expression of cell membrane protein claudin-5 and tight junction protein ZO-1. Element 1, element 5, and element 7 are the sites where HIF-1α interacts with MMP-9 at the transcription level. Conclusion This study shows that HIF-1α can increase the permeability of monolayer epithelial cells by inducing the high expression of MMP-9, leading to capillary leakage. Its target is at the −3798 bp, −1878 bp, and −1489 bp points of the transcription initiation site in the MMP-9 promoter region.

PFAS & Pesticides effect on cell Survival, Permeability and Tight Junction in an in-vitro model of the Human Blood-Brain Barrier

PFAS & Pesticides effect on cell Survival, Permeability and Tight Junction in an in-vitro model of the Human Blood-Brain Barrier

3,5,7,3',4'-Pentamethoxyflavone Enhances the Barrier Function through Transcriptional Regulation of the Tight Junction in Human Intestinal Caco-2 Cells.

The intestinal tight junction (TJ) barrier plays a pivotal role in the regulation of intestinal homeostasis. This study investigated the effects of 3,5,7,3',4'-pentamethoxyflavone (PMF), a major polymethoxyflavone found in black ginger, on TJ barrier regulation using intestinal Caco-2 cells. PMF treatment enhanced the TJ barrier integrity in Caco-2 cells, indicated by increased transepithelial electrical resistance (control, 1261 ± 36 Ω·cm2; 100 μM PMF, 1383 ± 55 Ω·cm2 at 48 h, p < 0.05) and decreased permeability to fluorescein-conjugated dextran (control, 24.2 ± 1.8 pmol/(cm2 × h); 100 μM PMF, 18.6 ± 1.0 pmol/(cm2 × h), p < 0.05). Immunoblot analysis revealed that PMF increased the cytoskeletal association and cellular expression of the TJ proteins, zonula occludens-1, claudin-3, and claudin-4 (e.g., occludin; control, 1.00 ± 0.2; 100 μM PMF, 3.69 ± 0.86 at 48 h, p < 0.05). Quantitative reverse transcriptase-polymerase chain reaction analysis and a luciferase promoter assay showed that PMF enhanced the transcription of occludin, claudin-3, and claudin-4. The promoter assay with site-directed mutagenesis indicated that PMF-induced occludin and claudin-3 transcription was mediated by transcription factors, KLF5 and EGR1, respectively, while PMF activated claudin-4 transcription through GATA1 and AP1. Taken together, the transcriptional regulation of TJ proteins is involved in PMF-mediated promotion of the intestinal barrier in vitro.

Inflammatory resolution and vascular barrier restoration after retinal ischemia reperfusion injury

BackgroundSeveral retinal pathologies exhibit both inflammation and breakdown of the inner blood-retinal barrier (iBRB) resulting in vascular permeability, suggesting that treatments that trigger resolution of inflammation may also promote iBRB restoration.MethodsUsing the mouse retinal ischemia-reperfusion (IR) injury model, we followed the time course of neurodegeneration, inflammation, and iBRB disruption and repair to examine the relationship between resolution of inflammation and iBRB restoration and to determine if minocycline, a tetracycline derivative shown to reverse microglial activation, can hasten these processes.ResultsA 90-min ischemic insult followed by reperfusion in the retina induced cell apoptosis and inner retina thinning that progressed for approximately 2 weeks. IR increased vascular permeability within hours, which resolved between 3 and 4 weeks after injury. Increased vascular permeability coincided with alteration and loss of endothelial cell tight junction (TJ) protein content and disorganization of TJ protein complexes. Shunting of blood flow away from leaky vessels and dropout of leaky capillaries were eliminated as possible mechanisms for restoring the iBRB. Repletion of TJ protein contents occurred within 2 days after injury, long before restoration of the iBRB. In contrast, the eventual re-organization of TJ complexes at the cell border coincided with restoration of the barrier. A robust inflammatory response was evident a 1 day after IR and progressed to resolution over the 4-week time course. The inflammatory response included a rapid and transient infiltration of granulocytes and Ly6C+ classical inflammatory monocytes, a slow accumulation of Ly6Cneg monocyte/macrophages, and activation, proliferation, and mobilization of resident microglia. Extravasation of the majority of CD45+ leukocytes occurred from the superficial plexus. The presence of monocyte/macrophages and increased numbers of microglia were sustained until the iBRB was eventually restored. Intervention with minocycline to reverse microglial activation at 1 week after injury promoted early restoration of the iBRB coinciding with decreased expression of mRNAs for the microglial M1 markers TNF-α, IL-1β, and Ptgs2 (Cox-2) and increased expression of secreted serine protease inhibitor Serpina3n mRNA.ConclusionsThese results suggest that iBRB restoration occurs as TJ complexes are reorganized and that resolution of inflammation and restoration of the iBRB following retinal IR injury are functionally linked.

Role of Neuroinflammation and Blood-Brain Barrier Permutability on Migraine

Currently, migraine is treated mainly by targeting calcitonin gene-related peptides, although the efficacy of this method is limited and new treatment strategies are desired. Neuroinflammation has been implicated in the pathogenesis of migraine. In patients with migraine, peripheral levels of pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α, are known to be increased. Additionally, animal models of headache have demonstrated that immunological responses associated with cytokines are involved in the pathogenesis of migraine. Furthermore, these inflammatory mediators might alter the function of tight junctions in brain vascular endothelial cells in animal models, but not in human patients. Based on clinical findings showing elevated IL-1β, and experimental findings involving IL-1β and both the peripheral trigeminal ganglion and central trigeminal vascular pathways, regulation of the Il-1β/IL-1 receptor type 1 axis might lead to new treatments for migraine. However, the integrity of the blood-brain barrier is not expected to be affected during attacks in patients with migraine.