Fluorescein Isothiocyanate-dextran Permeability Research Articles

Glucosamine-Mediated Hexosamine Biosynthesis Pathway Activation Uses ATF4 to Promote "Exercise-Like" Angiogenesis and Perfusion Recovery in PAD.

Endothelial cells (ECs) use glycolysis to produce energy. In preclinical models of peripheral arterial disease, further activation of EC glycolysis was ineffective or deleterious in promoting hypoxia-dependent angiogenesis, whereas pentose phosphate pathway activation was effective. Hexosamine biosynthesis pathway, pentose phosphate pathway, and glycolysis are closely linked. Glucosamine directly activates hexosamine biosynthesis pathway. Hind-limb ischemia in endothelial nitric oxide synthase knockout (eNOS-/-) and BALB/c mice was used. Glucosamine (600 μg/g per day) was injected intraperitoneally. Blood flow recovery was assessed using laser Doppler perfusion imaging and angiogenesis was studied by CD31 immunostaining. In vitro, human umbilical vein ECs and mouse microvascular ECs with glucosamine, L-glucose, or vascular endothelial growth factor (VEGF165a) were tested under hypoxia and serum starvation. Cell Counting Kit-8, tube formation, intracellular reactive oxygen species, electric cell-substrate impedance sensing, and fluorescein isothiocyanate dextran permeability were assessed. Glycolysis and oxidative phosphorylation were assessed by seahorse assay. Gene expression was assessed using RNA sequencing, real-time quantitative polymerase chain reaction, and Western blot. Human muscle biopsies from patients with peripheral arterial disease were assessed for EC O-GlcNAcylation before and after supervised exercise versus standard medical care. On day 3 after hind-limb ischemia, glucosamine-treated versus control eNOS-/- mice had less necrosis (n=4 or 5 per group). Beginning on day 7 after hind-limb ischemia, glucosamine-treated versus control BALB/c mice had higher blood flow, which persisted to day 21, when ischemic muscles showed greater CD31 staining per muscle fiber (n=8 per group). In vitro, glucosamine versus L-glucose ECs showed improved survival (n=6 per group) and tube formation (n=6 per group). RNA sequencing of glucosamine versus L-glucose ECs showed increased amino acid metabolism (n=3 per group). That resulted in increased oxidative phosphorylation (n=8-12 per group) and serine biosynthesis pathway without an increase in glycolysis or pentose phosphate pathway genes (n=6 per group). This was associated with better barrier function (n=6-8 per group) and less reactive oxygen species (n=7 or 8 per group) compared with activating glycolysis by VEGF165a. These effects were mediated by activating transcription factor 4, a driver of exercise-induced angiogenesis. In muscle biopsies from humans with peripheral arterial disease, EC/O-GlcNAcylation was increased by 12 weeks of supervised exercise versus standard medical care (n=6 per group). In cells, mice, and humans, activation of hexosamine biosynthesis pathway by glucosamine in peripheral arterial disease induces an "exercise-like" angiogenesis and offers a promising novel therapeutic pathway to treat this challenging disorder.

Jagged1-Notch1 Signaling Pathway Induces M1 Microglia to Disrupt the Barrier Function of Retinal Microvascular Endothelial Cells

Purpose Microglia-related inflammation is closely linked to the pathogenesis of retinal diseases. The primary objective of this research was to investigate the impact and mechanism of M1 phenotype microglia on the barrier function of retina microvascular endothelial cells. Methods Quantitative polymerase chain reactions and western blot techniques were utilized to analysis the mRNA and protein expressions of M1 and M2 markers of human microglial clone 3 cell line (HMC3), as well as the levels of Notch ligands and receptors under the intervention of lipopolysaccharide (LPS) or interleukin (IL)-4. ELISA was utilized to detect the pro-inflammatory and anti-inflammatory cytokines from HMC3 cells. The cellular tight junction and apoptosis of human retinal microvascular endothelial cells (HRMECs) were assessed by western blot and fluorescein isothiocyanate-dextran permeability assay. The inhibitors of Notch1 and RNA interference (RNAi) targeting Jagged1 were used to assess their contribution to the barrier function of vascular endothelial cells. Results Inducible nitric oxide synthase (iNOS) and IL-1β were considerably elevated in LPS-treated HMC3, while CD206 and Arg-1 markedly elevated under IL-4 stimulation. The conditioned medium derived from LPS-treated HMC3 cells promoted permeability, diminished the expression of zonula occludens-1 and Occludin, and elevated the expression of Cleaved caspase-3 in HRMECs. RNAi targeting Jagged1 or Notch1 inhibitor could block M1 HMC3 polarization and maintain barrier function of HRMECs. Conclusion Our findings suggest that Jagged1-Notch1 signaling pathway induces M1 microglial cells to disrupt the barrier function of HRMECs, which may lead to retinal diseases.

Intestinal epithelial barrier protective effect of nut oils (walnut, almond, pistachio, and pine nut) on TNF-α/IFN-γ induced damage in Caco-2 cell monolayers

Damage to the intestinal barrier causes intestinal inflammation, which could lead to the development of gastrointestinal diseases. Nut oils contain an abundance of unsaturated fatty acids, which are responsible for most of the bioactivities of nut oils. Here, the protective effect of four nut oils on the intestinal epithelial barrier was confirmed using Caco-2 cells damaged by tumor necrosis factor-α/interferon-γ treatment. Nut oils effectively increased transepithelial electronic resistance and lowered paracellular fluorescein isothiocyanate-dextran permeability in damaged Caco-2 cells. Furthermore, the nut oils inhibited the pro-inflammatory cytokine production of interleukin-6, which was induced by the inflammatory response. In particular, walnut oil effectively improved tight junction protein (occludin and zonula occludens-1) expression and distribution in damaged Caco-2 cells. In conclusion, our findings suggest that nut oils are promising candidates for impaired intestinal barrier protection.

Effect of Polycan, a β-Glucan from Aureobasidium pullulans SM-2001, on Inflammatory Response and Intestinal Barrier Function in DSS-Induced Ulcerative Colitis.

Ulcerative colitis (UC), a subtype of inflammatory bowel disease, is a chronic gastrointestinal inflammatory disease with unclear etiology and pathophysiology. Herein, we determined the effects of extracellular polysaccharides purified from Aureobasidium pullulans SM-2001 (Polycan) on tight junction protein expression, inflammation, and apoptosis in a dextran sodium sulfate (DSS)-induced acute colitis model. Fifty mice were divided into normal, DSS, DSS + Polycan 250 mg/kg (Polycan 250), DSS + Polycan 500 mg/kg (Polycan 500), and DSS + 5-aminosalicylic acid 100 mg/kg (5-ASA) groups. Their body weights, colon lengths, histological changes in colon tissue, and tight junction function were observed. Results showed that Polycan 250, Polycan 500, and 5-ASA significantly inhibited body weight loss compared with DSS. Similar to 5-ASA, Polycan 500 exhibited preventive effects on colon length shortening and histological changes in colon tissues. Polycan inhibited the DSS-induced decrease in fluorescein isothiocyanate-dextran permeability and myeloperoxidase activity. Moreover, Polycan significantly recovered serum cytokine (e.g., tumor necrosis factor-α, interleukin (IL)-6, and IL-1β) or mRNA expression in colon tissue compared with DSS. Polycan also inhibited apoptosis by reducing caspase-3 activity and the Bcl-2 associated X/B-cell lymphoma 2 (Bcl-2) ratio. Additionally, DSS treatment significantly reduced microbial abundance and diversity, but the administration of Polycan reversed this effect. Collectively, Polycan protected intestinal barrier function and inhibited inflammation and apoptosis in DSS-induced colitis.

The effects of astragaloside IV on gut microbiota and serum metabolism in a mice model of intracerebral hemorrhage

BackgroundAstragaloside IV (AS-IV) is the main active component of “Astragalus membranaceus (Fisch.) Bunge, a synonym of Astragalus propinquus Schischkin (Fabaceae)”, which demonstrated to be useful for the treatment of intracerebral hemorrhage (ICH). However, due to the low bioavailability and barrier permeability of AS-IV, the gut microbiota may be an important key regulator for AS-IV to work. ObjectiveTo explore the influences of gut microbiota on the effects of AS-IV on ICH. MethodsMice were randomly divided into five groups: sham, ICH, and AS-IV-treated groups (25 mg/kg, 50 mg/kg, and 100 mg/kg). Behavioral tests, brain histopathology, and immunohistochemistry analysis were used to evaluate the degree of brain injury. Western blot was employed to verify peri‑hematoma inflammation. The plasma lipopolysaccharide (LPS) leakage, the fluorescein isothiocyanate-dextran permeability, the colonic histopathology, and immunohistochemistry were detected to evaluate the barrier function of intestinal mucosal. Moreover, 16S rDNA sequencing and metabolomic analysis was applied to screen differential bacteria and metabolites, respectively. The correlation analysis was adopted to determine the potential relationship between differential bacteria and critical metabolites or neurological deficits. ResultsAS-IV alleviated neurological deficits, neuronal injury and apoptosis, and blood-brain barrier disruption. This compound reduced tumor necrosis factor (TNF)-α expression, increased arginase (Arg)-1 and interleukin (IL)-33 levels around the hematoma. Next, 16S rRNA sequencing indicated that AS-IV altered the gut microbiota, and inhibited the production of conditional pathogenic bacteria. Metabolomic analysis demonstrated that AS-IV regulated the serum metabolic profiles, especially the aminoacid metabolism and peroxisome proliferator-activated receptor (PPAR) signaling pathway. Additionally, AS-IV mitigated intestinal barrier damage and LPS leakage. ConclusionThis study provides a new perspective on the use of AS-IV for the treatment of ICH. Among them, gut microbiota and its metabolites may be the key regulator of AS-IV in treating ICH.

Lactiplantibacillus plantarum OLL2712 Induces Autophagy via MYD88 and Strengthens Tight Junction Integrity to Promote the Barrier Function in Intestinal Epithelial Cells.

Autophagy is an important system conserved in eukaryotes that maintains homeostasis by degrading abnormal proteins. Autophagy incompetence in intestinal epithelial cells causes the abnormal function of intestinal stem cells and other cells and damages intestinal barrier function. The disruption of the intestinal barrier causes chronic inflammation throughout the body, followed by impaired glucose and lipid metabolism. Lactiplantibacillus plantarum OLL2712 (OLL2712) is a lactic acid bacterium that induces interleukin-10 production from immune cells, alleviates chronic inflammation, and improves glucose and lipid metabolism. In this study, we hypothesized that OLL2712 exerts anti-inflammatory effects by inducing autophagy and ameliorating intestinal barrier dysfunction, and we investigated its autophagy-inducing activities and functions. Caco-2 cells stimulated with OLL2712 for 24 h showed an increased number of autolysosomes per cell, compared with unstimulated cells. Therefore, the permeability of fluorescein isothiocyanate dextran 4000 (FD-4) was suppressed by inducing autophagy. In contrast, mucin secretion in HT-29-MTX-E12 cells was also increased by OLL2712 but not via autophagy induction. Finally, the signaling pathway involved in autophagy induction by OLL2712 was found to be mediated by myeloid differentiation factor 88 (MYD88). In conclusion, our findings suggest that OLL2712 induces autophagy in intestinal epithelial cells via MYD88, and that mucosal barrier function is strengthened by inducing autophagy.

Siponimod as a novel inhibitor of retinal angiogenesis: in vitro and in vivo evidence of therapeutic efficacy.

S1P receptors control endothelial cell proliferation, migration, and survival. Evidence of the ability of S1P receptor modulators to influence multiple endothelial cell functions suggests their potential use for antiangiogenic effect. The main purpose of our study was to investigate the potential of siponimod for the inhibition of ocular angiogenesis in vitro and in vivo We investigated the effects of siponimod on the metabolic activity (MTT assay), cell toxicity (LDH release), basal proliferation and growth factor induced proliferation (BrdU assay), and migration (transwell migration assay) of human umbilical vein endothelial cells (HUVEC) and retinal microvascular endothelial cells (HRMEC). The effects of siponimod on HRMEC monolayer integrity, and barrier function under basal conditions and TNF-a induced disruption were assessed using the trans-endothelial electrical resistance (TEER) and FITC-dextran permeability assays. Siponimod's effect on TNF-α induced distribution of barrier proteins in HRMEC was investigated using immunofluorescence. Finally, the effect of siponimod on ocular neovascularization in vivo was assessed using suture-induced corneal neovascularization in albino rabbits. Our results show that siponimod did not affect endothelial cell proliferation or metabolic activity, but significantly inhibited endothelial cell migration, increased HRMEC barrier integrity, and reduced TNF-a induced barrier disruption. Siponimod also protected against TNF-a induced disruption of claudin-5, ZO-1, and VE-cadherin in HRMEC. These actions are mainly mediated by S1PR1 modulation. Finally, siponimod prevented the progression of suture-induced corneal neovascularization in albino rabbits. In conclusion, the effects of siponimod on various processes known to be involved in angiogenesis support its therapeutic potential in disorders associated with ocular neovascularization. Significance Statement Siponimod is an extensively characterized S1P receptor modulator already approved for the treatment of multiple sclerosis. It inhibits retinal endothelial cell migration, potentiates endothelial barrier function, protects against TNF-a induced barrier disruption, and also inhibits suture-induced corneal neovascularization in rabbits. These results support its use for a novel therapeutic indication in the management of ocular neovascular diseases.

Yogurt starter strains ameliorate intestinal barrier dysfunction via activating AMPK in Caco-2 cells

ABSTRACT Lactic acid bacteria (LAB) are commonly used probiotics that improve human health in various aspects. We previously reported that yogurt starter strains, Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, potentially enhance the intestinal epithelial barrier function by inducing the expression of antimicrobial peptides in the small intestine. However, their effects on physical barrier functions remain unknown. In this study, we found that both strains ameliorated the decreased trans-epithelial resistance and the increased permeability of fluorescein isothiocyanate-dextran induced by tumor necrosis factor (TNF)-α and interferon (IFN)-γ in Caco-2 cells. We also demonstrated that LAB prevented a decrease in the expression and disassembly of tight junctions (TJs) induced by TNF-α and IFN-γ. To assess the repair activity of TJs, a calcium switch assay was performed. Both strains were found to promote the reassembly of TJs, and their activity was canceled by the inhibitor of AMP-activated protein kinase (AMPK). Moreover, these strains showed increased AMPK phosphorylation. These observations suggest that the strains ameliorated physical barrier dysfunction via the activation of AMPK. The activities preventing barrier destruction induced by TNF-α and IFN-γ were strain-dependent. Several strains containing L. bulgaricus 2038 and S. thermophilus 1131 significantly suppressed the barrier impairment, and L. bulgaricus 2038 showed the strongest activity among them. Our findings suggest that the intake of L. bulgaricus 2038 and S. thermophilus 1131 is a potential strategy for the prevention and repair of leaky gut.

PPARγ activation inhibits endocytosis of claudin-4 and protects against deoxynivalenol-induced intestinal barrier dysfunction in IPEC-J2 cells and weaned piglets

The role of peroxisome proliferator activated receptor gamma (PPARγ) in the regulation of adipocyte differentiation has been well characterized. Besides adipose tissue, PPARγ is also highly expressed in the intestine. However, the functional role of PPARγ in the regulation of intestinal function still remains poorly understood. In the present study, we sought to understand the role of PPARγ activation on regulation of intestinal barrier function in intestinal porcine epithelial cells (IPEC-J2) and weaned piglets exposed to the mycotoxin, deoxynivalenol (DON). PPARγ activation by rosiglitazone and troglitazone, two pharmacological PPARγ ligands, increased the protein expression of tight junction proteins (TJP), claudin-3 and 4. PPARγ inhibition increased endocytosis of claudin-4 which was reversed by its activation with troglitazone. DON exposure decreased the protein expression of TJP, and also significantly suppressed PPARγ transcriptional activity. Interestingly, PPARγ activation reversed the reduction of claudin-3 and 4 caused by DON in vitro and in vivo. PPARγ activation also partially restored the transepithelial electrical resistance (TEER) and reduced the permeability of fluorescein isothiocyanate-dextran (FITC-dextran) that have been negatively impacted by DON. These effects were lost in the presence of a specific PPARγ antagonist or in PPARγ knockout cells, confirming the importance of PPARγ in the regulation of intestinal barrier function and integrity. Likewise, in weaned pigs exposed to DON, the PPARγ agonist pioglitazone mitigated the impaired villus-crypt morphology caused by DON. Therefore, pharmacological and natural bioactive compounds with PPARγ stimulatory activities could be effective in preventing DON-induced gut barrier dysfunction.

NE-activated β2-AR/β-arrestin 2/Src pathway mediates duodenal hyperpermeability induced by water-immersion restraint stress.

Stress causes a rapid spike in norepinephrine (NE) levels, leading to gastrointestinal dysfunction. NE reduces the expression of tight junctions (TJs) and aggravates intestinal mucosal damage, but the regulatory mechanism is still unclear. The present study aimed to investigate the molecular mechanisms underlying the regulation of stress-associated duodenal hyperpermeability by NE. Fluorescein isothiocyanate-dextran permeability, transepithelial resistance, immunofluorescence, Western blot, and high-performance liquid chromatography analysis were used in water-immersion restraint stress (WIRS) rats in this study. The results indicate that the duodenal permeability, degradation of TJs, mucosal NE, and β2-adrenergic receptor (β2-AR) increased in WIRS rats. The duodenal intracellular cyclic adenosine monophosphate levels were decreased, whereas the expression of β-arrestin 2 negatively regulates G protein-coupled receptors signaling, was significantly increased. Src recruitment was mediated by β-arrestin; thus, the levels of Src kinase activation were enhanced in WIRS rats. NE depletion, β2-AR, or β-arrestin 2 blockade significantly decreased mucosal permeability and increased TJs expression, suggesting improved mucosal barrier function. Moreover, NE induced an increased duodenal permeability of normal rats with activated β-arrestin 2/Src signaling, which was significantly inhibited by β2-AR blockade. The present findings demonstrate that the enhanced NE induced an increased duodenal permeability in WIRS rats through the activated β2-AR/β-arrestin 2/Src pathway. This study provides novel insight into the molecular mechanism underlying the regulation of NE on the duodenal mucosal barrier and a new target for treating duodenal ulcers induced by stress.

Investigation of murine host sex as a biological variable in epithelial barrier function and muscle contractility in human intestinal organoids.

Intestinal failure (IF) occurs when intestinal surface area or function is not sufficient to support digestion and nutrient absorption. Human intestinal organoid (HIO)-derived tissue-engineered intestine is a potential cure for IF. Research to date has demonstrated successful HIO transplantation (tHIO) into mice with significant in vivo maturation. An area lacking in the literature is exploration of murine host sex as a biological variable (SABV) in tHIO function. In this study, we investigate murine host SABV in tHIO epithelial barrier function and muscle contractility. HIOs were generated in vitro and transplanted into nonobese diabetic, severe combined immunodeficiency gamma chain deficient male and female mice. tHIOs were harvested after 8-12 weeks in vivo. Reverse transcriptase polymerase chain reaction and immunohistochemistry were conducted to compare tight junctions and contractility-related markers in tHIOs. An Ussing chamber and contractility apparatus were used to evaluate tHIO epithelial barrier and muscle contractile function, respectively. The expression and morphology of tight junction and contractility-related markers from tHIOs in male and female murine hosts is not significantly different. Epithelial barrier function as measured by transepithelial resistance, short circuit current, and fluorescein isothiocyanate-dextran permeability is no different in tHIOs from male and female hosts, although these results may be limited by HIO epithelial immaturity and a short flux time. Muscle contractility as measured by total contractile activity, amplitude, frequency, and tension is not significantly different in tHIOs from male and female hosts. The data suggest that murine host sex may not be a significant biological variable influencing tHIO function, specifically epithelial barrier maintenance and muscle contractility, though limitations exist in our model.

Novel Histone Deacetylase 6 Inhibitor Confers Anti-inflammatory Effects and Enhances Gut Barrier Function.

The purpose of the current study was to examine the anti-inflammatory effects of CKD-506, a novel histone deacetylase 6 inhibitor, on human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells and to explore the relationship between CKD-506 and gut epithelial barrier function. Lipopolysaccharide-stimulated human PBMCs from inflammatory bowel disease (IBD) patients were treated with CKD-506, and tumor necrosis factor (TNF)-α expression was measured using an enzyme-linked immunosorbent assay. The proliferation of CD4+ T cells from IBD patients was evaluated using flow cytometric analysis. The effects of CKD-506 on gut barrier function in a cell line and colon organoids, based on examinations of mRNA production, goblet cell differentiation, and E-cadherin recovery, were investigated using quantitative reverse transcription polymerase chain reaction, immunofluorescence, and a fluorescein isothiocyanate-dextran permeability assay. Secretion of TNF-α, a pivotal pro-inflammatory mediator in IBD, by lipopolysaccharide-triggered PBMCs was markedly decreased by CKD-506 treatment in a dose-dependent manner and to a greater extent than by tofacitinib or tubastatin A treatment. E-cadherin mRNA expression and goblet cell differentiation increased significantly and dose-dependently in HT-29 cells in response to CKD-506, and inhibition of E-cadherin loss after TNF-α stimulation was significantly reduced both in HT-29 cells and gut organoids. Caco-2 cells treated with CKD-506 showed a significant reduction in barrier permeability in a dose-dependent manner. The present study demonstrated that CKD-506 has anti-inflammatory effects on PBMCs and CD4 T cells and improves gut barrier function, suggesting its potential as a small-molecule therapeutic option for IBD.

All-trans Retinoic Acids Synergistically and Beneficially Affect In Vitro Glaucomatous Trabecular Meshwork (TM) Models Using 2D and 3D Cell Cultures of Human TM Cells

We report herein on the effects of all-trans retinoic acid (ATRA) on two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork (HTM) cells that were treated with transforming growth factor β2 (TGF-β2). In the presence of 5 ng/mL TGF-β2, the effects of ATRA on the following were observed: (1) the barrier function of the 2D HTM monolayers, as determined by trans-endothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) dextran permeability measurements; (2) a Seahorse cellular bio-metabolism analysis; (3) physical properties, including the size and stiffness, of 3D spheroids; (4) the gene expression of extracellular matrix (ECM) molecules, ECM modulators including tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), tight junction (TJ)-related molecules, and endoplasmic reticulum (ER)-stress-related factors. ATRA significantly inhibited the TGF-β2-induced increase in the TEER values and FITC dextran permeability of the 2D monolayers, while an ATRA monotreatment induced similar effects as TGF-β2. A real-time metabolic analysis revealed that ATRA significantly inhibited the TGF-β2-induced shift in metabolic reserve from mitochondrial oxidative phosphorylation to glycolysis in 2D HTM cells, whereas ATRA alone did not induce significant metabolic changes. In contrast, ATRA induced the formation of substantially downsized and softer 3D spheroids in the absence and presence of TGF-β2. The different effects induced by ATRA toward 2D and 3D HTM cells were also supported by the qPCR analysis of several proteins as above. The findings reported here indicate that ATRA may induce synergistic and beneficial effects on TGF-β2-treated 2D- and 3D-cultured HTM cells; those effects varied significantly between the 2D and 3D cultures.

Capsaicin Attenuates Lipopolysaccharide-Induced Inflammation and Barrier Dysfunction in Intestinal Porcine Epithelial Cell Line-J2.

Capsaicin is a spicy, highly pungent, colorless, vanilloid compound found in chili peppers with anti-inflammatory, antioxidant, anti-cancer, and analgesic properties. However, the protective effects of capsaicin on the pig intestine during inflammation are yet to be explored. This study investigated the effects of capsaicin on the gut inflammatory response, intestinal epithelial integrity, and gene expression level of nutrient transporters in a model of lipopolysaccharide (LPS)-induced inflammation in non-differentiated intestinal porcine epithelial cell line-J2 (IPEC-J2). The results showed that the pre-treatment of cells with capsaicin (100 μM) significantly decreased the gene expression and secretion of proinflammatory cytokines induced by LPS through Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. In addition, pre-treatment of cells with capsaicin also increased both gene and protein abundance of tight junction proteins. Furthermore, pre-treatment cells with capsaicin significantly increased trans-epithelial electrical resistance (TEER) and decreased permeability of fluorescein isothiocyanate-dextran (FD4) from the apical side to the basolateral side compared with the control (P < 0.05). Additionally, pre-treatment of cells with capsaicin upregulated the mRNA abundance of nutrients transporters such as Na+/glucose cotransporter 1 (SGLT1). These results suggested that capsaicin could attenuate LPS-induced inflammation response through TLR4/NF-κB pathway and improve barrier integrity and glucose absorption.

Screening of the Drug-Induced Effects of Prostaglandin EP2 and FP Agonists on 3D Cultures of Dexamethasone-Treated Human Trabecular Meshwork Cells.

The objective of the current study was to perform a screening of the drug-induced effects of the prostaglandin F2α (PGF2α) and EP2 agonist, omidenepag (OMD), using two- and three-dimensional (2D and 3D) cultures of dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells. The drug-induced effects on 2D monolayers were characterized by measuring the transendothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)–dextran permeability, the physical properties of 3D spheroids, and the gene expression of extracellular matrix (ECM) molecules, including collagen (COL) 1, 4 and 6, and fibronectin (FN), α smooth muscle actin (αSMA), a tissue inhibitor of metalloproteinase (TIMP) 1–4, matrix metalloproteinase (MMP) 2, 9 and 14 and endoplasmic reticulum (ER) stress-related factors. DEX induced a significant increase in TEER values and a decrease in FITC–dextran permeability, respectively, in the 2D HTM monolayers, and these effects were substantially inhibited by PGF2α and OMD. Similarly, DEX also caused decreased sizes and an increased stiffness in the 3D HTM spheroids, but PGF2α or OMD had no effects on the stiffness of the spheroids. Upon exposure to DEX, the following changes were observed: the upregulation of COL4 (2D), αSMA (2D), and TIMP4 (2D and 3D) and the downregulation of TIMP1 and 2 (3D), MMP2 and 14 (3D), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) (2D), and glucose regulator protein (GRP)78 (3D). In the presence of PGF2α or OMD, the downregulation of COL4 (2D), FN (3D), αSMA (2D), TIMP3 (3D), MMP9 (3D) and the CCAAT/enhancer-binding protein homologous protein (CHOP) (2D), and the upregulation of TIMP4 (2D and 3D), MMP2, 9 and 14 (2D), respectively, were observed. The findings presented herein suggest that 2D and 3D cell cultures can be useful in screening for the drug-induced effects of PGF2α and OMD toward DEX-treated HTM cells.

Urban Particulate Matters May Affect Endoplasmic Reticulum Stress and Tight Junction Disruption in Nasal Epithelial Cells.

Exposure to airborne urban particulate matter (UPM) has been closely related to the development and aggravation of respiratory disease, including sinonasal disorders. The aims of this study were to investigate the effect of UPM on nasal epithelial tight junctions (TJs) and mucosal barrier function and delineate the underlying mechanism by using both in vitro and in vivo models. In this study, human nasal epithelial cells (hNECs) and BALB/c mice were exposed to UPMs. UPM 1648a and 1649 b were employed. TJ and endoplasmic reticulum (ER) stress marker expression was measured using western blot analysis and immunofluorescence. TJ integrity and nasal epithelial barrier function were evaluated by transepithelial electric resistance (TER) and paracellular flux. In addition, the effects of N-acetyl-L-cysteine (NAC) on UPM-induced nasal epithelial cells were investigated. UPM significantly impaired the nasal epithelial barrier, as demonstrated by decreased protein expression of TJ and ER stress markers in human nasal epithelial cells. This finding was in parallel to reduced transepithelial electrical resistance and increased fluorescein isothiocyanate-dextran permeability. Pretreatment with NAC decreased the degree of UPM-mediated ER stress and restored nasal epithelial barrier disruption in human nasal epithelial cells (hNEC) and the nasal mucosa of experimental animals. These data suggest that UPMs may induce nasal epithelial barrier dysfunction by targeting TJs and ER stress could be related in this process. Based on these results, we suggest that suppression of this process with an inhibitor targeting ER stress responses could represent a novel promising therapeutic target in UPM-induced sinonasal disease.

Aflatoxin B1 and Aflatoxin M1 Induce Compromised Intestinal Integrity through Clathrin-Mediated Endocytosis.

With the growing diversity and complexity of diet, humans are at risk of simultaneous exposure to aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1), which are well-known contaminants in dairy and other agricultural products worldwide. The intestine represents the first barrier against external contaminants; however, evidence about the combined effect of AFB1 and AFM1 on intestinal integrity is lacking. In vivo, the serum biochemical parameters related to intestinal barrier function, ratio of villus height/crypt depth, and distribution pattern of claudin-1 and zonula occluden-1 were significantly affected in mice exposed to 0.3 mg/kg b.w. AFB1 and 3.0 mg/kg b.w. AFM1. In vitro results on differentiated Caco-2 cells showed that individual and combined AFB1 (0.5 and 4 μg/mL) and AFM1 (0.5 and 4 μg/mL) decreased cell viability and trans-epithelial electrical resistance values as well as increased paracellular permeability of fluorescein isothiocyanate-dextran in a dose-dependent manner. Furthermore, AFM1 aggravated AFB1-induced compromised intestinal barrier, as demonstrated by the down-regulation of tight junction proteins and their redistribution, particularly internalization. Adding the inhibitor chlorpromazine illustrated that clathrin-mediated endocytosis partially contributed to the compromised intestinal integrity. Synergistic and additive effects were the predominant interactions, suggesting that these toxins are likely to have negative effects on human health.

Fusobacterium nucleatum exacerbates colitis by damaging epithelial barriers and inducing aberrant inflammation.

Fusobacterium nucleatum (F. nucleatum) has been reported to be enriched in patients with inflammatory bowel disease (IBD). This study aimed to explore the role of F. nucleatum in IBD and its pathogenic mechanism. Several bacteria that have been reported to be associated with IBD or colorectal cancer were measured in the fecal samples of 91 patients with IBD and 43 healthy individuals. Mice with dextran sulfate sodium (DSS)-induced colitis and a Caco-2 cell line were used to explore the pathogenicity of F. nucleatum. Barrier damage was evaluated by a transmission electron microscope, the permeability of fluorescein isothiocyanate-dextran, transepithelial electrical resistance and immunofluorescence. Protein levels of the cell-cell junction and activation of the STAT3 signaling pathway were detected by immunohistochemistry and immunoblot. Cytokine secretion and T-cell differentiation were measured by quantitative real-time polymerase chain reaction and flow cytometry. F. nucleatum was significantly enriched in the feces of patients with IBD and its abundance correlated with disease activity. Administration of F. nucleatum markedly exacerbated colitis in a DSS mouse model. Mechanistically, F. nucleatum damaged epithelial integrity and increased permeability by regulating the expression and distribution of tight junction proteins zonula occludens-1 and occludin. Moreover, F. nucleatum promoted the secretion of cytokines (tumor necrosis factor-α, interferon-γ, interleukin [IL]-1β, IL-6, and IL-17), activated the STAT3 signaling pathway, and induced CD4+ T cell proliferation and Th1 and Th17 subset differentiations. F. nucleatum can damage the intestinal barrier and induce aberrant inflammation, which exacerbates colitis.

Ion transport mechanisms for smoke inhalation-injured airway epithelial barrier.

Smoke inhalation injury is the leading cause of death in firefighters and victims. Inhaled hot air and toxic smoke are the predominant hazards to the respiratory epithelium. We aimed to analyze the effects of thermal stress and smoke aldehyde on the permeability of the airway epithelial barrier. Transepithelial resistance (RTE) and short-circuit current (ISC) of mouse tracheal epithelial monolayers were digitized by an Ussing chamber setup. Zonula occludens-1 tight junctions were visualized under confocal microscopy. A cell viability test and fluorescein isothiocyanate-dextran assay were performed. Thermal stress (40°C) decreased RTE in a two-phase manner. Meanwhile, thermal stress increased ISC followed by its decline. Na+ depletion, amiloride (an inhibitor for epithelial Na+ channels [ENaCs]), ouabain (a blocker for Na+/K+-ATPase), and CFTRinh-172 (a blocker of cystic fibrosis transmembrane regulator [CFTR]) altered the responses of RTE and ISC to thermal stress. Steady-state 40°C increased activity of ENaCs, Na+/K+-ATPase, and CFTR. Acrolein, one of the main oxidative unsaturated aldehydes in fire smoke, eliminated RTE and ISC. Na+ depletion, amiloride, ouabain, and CFTRinh-172 suppressed acrolein-sensitive ISC, but showed activating effects on acrolein-sensitive RTE. Thermal stress or acrolein disrupted zonula occludens-1 tight junctions, increased fluorescein isothiocyanate-dextran permeability but did not cause cell death or detachment. The synergistic effects of thermal stress and acrolein exacerbated the damage to monolayers. In conclusion, the paracellular pathway mediated by the tight junctions and the transcellular pathway mediated by active and passive ion transport pathways contribute to impairment of the airway epithelial barrier caused by thermal stress and acrolein. Graphical abstract Thermal stress and acrolein are two essential determinants for smoke inhalation injury, impairing airway epithelial barrier. Transcellular ion transport pathways via the ENaC, CFTR, and Na/K-ATPase are interrupted by both thermal stress and acrolein, one of the most potent smoke toxins. Heat and acrolein damage the integrity of the airway epithelium through suppressing and relocating the tight junctions.

Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity

BackgroundIn vitro blood–brain barrier (BBB) models using human induced pluripotent stem (iPS) cell-derived brain microvascular endothelial-like cells (iBMELCs) have been developed to predict the BBB permeability of drug candidates. For the differentiation of iBMELCs, Matrigel, which is a gelatinous protein mixture, is often used as a coating substrate. However, the components of Matrigel can vary among lots, as it is obtained from mouse sarcoma cells with the use of special technics and also contains various basement membranes. Therefore, fully defined substrates as substitutes for Matrigel are needed for a stable supply of iBMELCs with less variation among lots.MethodsiBMELCs were differentiated from human iPS cells on several matrices. The barrier integrity of iBMELCs was evaluated based on transendothelial electrical resistance (TEER) values and permeability of fluorescein isothiocyanate-dextran 4 kDa (FD4) and Lucifer yellow (LY). Characterization of iBMELCs was conducted by RT-qPCR and immunofluorescence analysis. Functions of efflux transporters were defined by intracellular accumulation of the substrates in the wells of multiwell plates.ResultsiBMELCs differentiated on laminin 221 fragment (LN221F-iBMELCs) had higher TEER values and lower permeability of LY and FD4 as compared with iBMELCs differentiated on Matrigel (Matrigel-iBMELCs). Besides, the gene and protein expression levels of brain microvascular endothelial cells (BMEC)-related markers were similar between LN221F-iBMELCs and Matrigel-iBMELCs. Moreover, both Matrigel- and LN221F-iBMELCs had functions of P-glycoprotein and breast cancer resistance protein, which are essential efflux transporters for barrier functions of the BBB.ConclusionThe fully defined substrate LN221F presents as an optimal coating matrix for differentiation of iBMELCs. The LN221F-iBMELCs had more robust barrier function for a longer period than Matrigel-iBMELCs with characteristics of BMECs. This finding will contribute the establishment of an iBMELC supply system for pharmacokinetic and pathological models of the BBB.