Fluorescein Isothiocyanate-conjugated Dextran Research Articles

Enhanced transscleral delivery using superficial ultrasound exposure and drug-loaded hydrogel

This study employed superficial ultrasound exposure of good ocular safety and a drug-loaded hydrogel of long residence time to enable transscleral delivery. First, we designed an acoustic adaptor to limit the ultrasound exposure depth to 1.59 mm to protect the posterior eye segments. Then, we optimized the alginate/polyacrylamide ratio (3:7) of a dual-crosslinked hydrogel to enable ultrasound-triggered release of model drug (70-kDa fluorescein isothiocyanate-conjugated dextran). Using fluorescence imaging to quantify the drug release, we showed that the developed method resulted in enhanced transscleral delivery in both ex vivo porcine scleras (2.6-fold) and in vivo rabbit scleras (2.2-fold). We also demonstrated that the method increased the drug penetration depth to the whole thickness of the sclera. In particular, the drug release efficiency increased with increasing ultrasound exposure time (1 and 3 min) and intensity (8, 19, 36, and 61 mW/cm2). Using scanning electron microscopy, we revealed that ultrasound exposure resulted in rougher surfaces and microscale rupture of the hydrogel. Moreover, Masson staining of scleral slices showed that the integrity of the top scleral fibers was disturbed by ultrasound exposure, and this disturbance recovered 3 days later. Our work demonstrates that the developed method holds great potential for mediating ocular drug delivery.

Pomegranate Extract Affects Gut Biofilm Forming Bacteria and Promotes Intestinal Mucosal Healing Regulating the Crosstalk between Epithelial Cells and Intestinal Fibroblasts

Background: Pomegranate (Punica granatum) can be used to prepare a bioactive extract exerting anti-inflammatory activities. Clinical studies demonstrated an improvement in clinical response in inflammatory bowel disease (IBD) patients when pomegranate extract (PG) was taken as a complement to standard medications. However, the molecular mechanisms underlying its beneficial effects are still scarcely investigated. This study investigates the effect of PG on bacterial biofilm formation and the promotion of mucosal wound healing. Methods: The acute colitis model was induced in C57BL/6N mice by 3% dextran sodium sulfate administration in drinking water for 5 days. During the recovery phase of colitis, mice received saline or PG (200 mg/kg body weight) by oral gavage for 11 days. Colitis was scored daily by evaluating body weight loss, bleeding, and stool consistency. In vivo intestinal permeability was evaluated by fluorescein isothiocyanate-conjugated dextran assay, bacterial translocation was assessed by fluorescence in situ hybridization on tissues, whereas epithelial and mucus integrity were monitored by immunostaining for JAM-A and MUC-2 markers. Bacterial biofilm formation was assessed using microfluidic devices for 24 or 48 h. Primary fibroblasts were isolated from healthy and inflamed areas of 8 IBD patients, and Caco-2 cells were stimulated with or without PG (5 μg/mL). Inflammatory mediators were measured at the mRNA and protein level by RT-PCR, WB, or Bio-plex multiplex immunoassay, respectively. Results: In vivo, PG boosted the recovery phase of colitis, promoting a complete restoration of the intestinal barrier with the regeneration of the mucus layer, as also demonstrated by the absence of bacterial spread into the mucosa and the enrichment of crypt-associated fibroblasts. Microfluidic experiments did not highlight a specific effect of PG on Enterobacterales biofilm formation, even though Citrobacter freundii biofilm was slightly impaired in the presence of PG. In vitro, inflamed fibroblasts responded to PG by downregulating the release of metalloproteinases, IL-6, and IL-8 and upregulating the levels of HGF. Caco-2 cells cultured in a medium supplemented with PG increased the expression of SOX-9 and CD44, whereas in the presence of HGF or plated with a fibroblast-conditioned medium, they displayed a decrease in SOX-9 and CD44 expression and an increase in AXIN2, a negative regulator of Wnt signaling. Conclusions: These data provide new insight into the manifold effects of PG on promoting mucosal homeostasis in IBD by affecting pathogen biofilm formation and favoring the regeneration of the intestinal barrier through the regulation of the crosstalk between epithelial and stromal cells.

P092 Punica Granatum affects gut biofilm-forming bacteria and promotes intestinal mucosal healing regulating the crosstalk between epithelial cells and intestinal fibroblasts

Abstract Background Punica Granatum (PG), is a bioactive compound of Pomegranate exerting anti-inflammatory activities. Clinical studies have demonstrated an improvement of clinical response in inflammatory bowel diseases (IBD) patients when PG was taken as a complement to standard medications. However, the molecular mechanisms underlying its beneficial effects are still scarcely investigated. In this study, we assessed the effects of PG on bacterial biofilm formation, and we investigated the properties of the PG to promote mucosal wound healing. Methods Bacterial biofilm formation was assessed using microfluidic devices for 24 or 48 hours. Acute colitis model was induced in C57BL/6N mice by 3% dextran sodium sulfate administration in their drinking water for 5 days. During the recovery phase of colitis, mice received saline or PG (200 mg/kg body weight) oral gavage for 11 days. . Colitis was scored daily evaluating the body weight loss, bleeding and consistency of stool. In vivo intestinal permeability was performed by fluorescein isothiocyanate-conjugated dextran assay, bacterial translocation by Fluorescence in situ hybridization on tissues, whereas epithelial and mucus integrity by immunostaining for JAM-A and MUC-2 markers. Primary fibroblasts, isolated from healthy and inflamed area of 6 IBD patients, and Caco-2 cells were stimulated with or without PG (15 ug/ml). Inflammatory mediators were measured at mRNA and protein levels by RT-PCR, WB or by Bio-plex multiplex immunoassay respectively. Results Microfluidic experiments revealed beneficial effects of PG to inhibit biofilm formation of GRAM- bacteria and to promote the expansion of some intestinal commensal Gram+ bacteria. In vivo, PG boosted the recovery phase of colitis promoting a complete restoration of intestinal barrier with the regeneration of the mucus layer compared to saline group as also demonstrated by no traces of bacteria spread into the mucosa. The PG enriched crypt-associated fibroblasts. In vitro, inflamed fibroblasts responded to PG by down-regulating the release of metalloproteinases, IL-6, IL-8 and up-regulating the levels of HGF. Caco-2 cells cultured in medium supplemented with PG increased the expression of Sox-9 and CD44, whereas in the presence of HGF or when plated with fibroblast-conditioned medium displayed a decrease expression of Sox-9 and CD44 and increase of AXIN2, a negative regulator of Wnt signaling. Conclusion These data provide new insight of the multiple effects of PG on promoting mucosal homeostasis in IBD by affecting biofilm formation of pathogens, promoting expansion of commensals and favoring the regeneration of intestinal barrier by regulating the crosstalk between epithelial cells and stromal cells.

Performance of Cell-Penetrating Peptides Anchored to Polysaccharide Platforms Applied via Various Mucosal Routes as an Absorption Enhancer.

We have been investigating the potential of cell-penetrating peptides anchored to polymeric platforms as a novel absorption enhancer which delivers biologics into systemic circulation via mucosal routes. Our previous mouse experiments demonstrated that hyaluronic acid modified with l-octaarginine, a typical cell-penetrating peptide, via a tetraglycine spacer significantly enhanced the mucosal absorption of protein drugs applied into the nasal cavities, irrespective of the molecular weights (Mw) of the drugs. The present study evaluated the performance of tetraglycine-l-octaarginine-linked hyaluronic acid applied via various mucosal routes. Somatropin (Mw: ca. 22.1 kDa) was moderately absorbed from the lung mucosa, and the mean absolute bioavailability (BA) reached 19% under enhancer-free conditions; nevertheless, its BA under intranasal administration was approximately 1% or less. Its BA significantly elevated to 46% on average through intrapulmonary coadministration with tetraglycine-l-octaarginine-linked hyaluronic acid. When the administration site was replaced with the oral cavities, an extreme reduction in somatropin absorption was observed with a mean BA of 0.056% under enhancer-free conditions. Intraoral coadministration with tetraglycine-l-octaarginine-linked hyaluronic acid resulted in a 6.3-fold elevation of somatropin absorption with statistical significance. A similar enhancement was observed under intrarectal administration with a further reduction in BA. On the other hand, the hyaluronic acid derivative did not exhibit the absorption-enhancing ability under intragastric administration, probably due to the lack of stabilization effects against enzyme-susceptible biologics. The results indicated that the intrapulmonary route was suitable for maximizing the mucosal absorption of biologics, and that there was a likelihood of the intraoral route with user convenience. When somatropin was substituted with fluorescein isothiocyanate-conjugated dextran with an average Mw range of 4-70 kDa, similar phenomena were observed under intrapulmonary and intranasal administration. BA decreased with an increase in the Mw of dextran; however, the ratio of BA under enhancer-present conditions to that under enhancer-free conditions was consistently around 3, indicating that the performance of the hyaluronic acid derivative was Mw-independent, irrespective of the administration route.

Highly Sensitive, Flow Cytometry-Based Measurement of Intestinal Permeability in Models of Experimental Colitis

Increased intestinal permeability is seen in a variety of inflammatory conditions such as enteric infections and inflammatory bowel disease. Because barrier function can provide a key biomarker of disease severity, it often is assayed in animal models. A common methodology involves gavaging mice with fluorescein isothiocyanate-conjugated dextran (FITC-D), followed by cardiac puncture to assay plasma fluorescence on a spectrophotometer. Although the FITC-D method is relatively simple, its sensitivity is limited and enables only a single measurement because the test requires killing the subject. Herein, we describe a novel flow cytometry-based method of intestinal permeability measurement based on detection of orally gavaged ovalbumin (OVA) that leaks out of the gut. Our approach uses minute blood volumes collected from the tail vein, permitting repeated testing of the same subject at multiple time points. By comparing this assay against the gold standard FITC-D method, we show the expanded utility of our OVA assay in measuring intestinal permeability. We directly compared our OVA assay against the FITC-D assay by co-administering both probes orally to the same animals and subsequently using their respective methodologies to measure intestinal permeability by detecting probe levels in the plasma. Permeability was assessed in mice genetically deficient in intestinal mucus production or glycosylation. In addition, wild-type mice undergoing dextran sodium sulfate-induced colitis or infected by the enteric bacterial pathogen Citrobacter rodentium also were tested. The OVA assay showed very high efficacy in all animal models of intestinal barrier dysfunction tested. Besides identifying intestinal barrier dysfunction in mice with impaired mucin glycosylation, the assay also allowed for repeated tracking of intestinal permeability within the same animal over time, providing data that cannot be easily acquired with other currently applied methods. The OVA assay is a highly sensitive and effective method of measuring intestinal permeability in mouse models of barrier dysfunction and experimental colitis.

Recombinant angiopoietin-like protein 4 attenuates intestinal barrier structure and function injury after ischemia/reperfusion.

BACKGROUNDIntestinal barrier breakdown, a frequent complication of intestinal ischemia-reperfusion (I/R) including dysfunction and the structure changes of the intestine, is characterized by a loss of tight junction and enhanced permeability of the intestinal barrier and increased mortality. To develop effective and novel therapeutics is important for the improvement of outcome of patients with intestinal barrier deterioration. Recombinant human angiopoietin-like protein 4 (rhANGPTL4) is reported to protect the blood-brain barrier when administered exogenously, and endogenous ANGPTL4 deficiency deteriorates radiation-induced intestinal injury. AIMTo identify whether rhANGPTL4 may protect intestinal barrier breakdown induced by I/R.METHODSIntestinal I/R injury was elicited through clamping the superior mesenteric artery for 60 min followed by 240 min reperfusion. Intestinal epithelial (Caco-2) cells and human umbilical vein endothelial cells were challenged by hypoxia/ reoxygenation to mimic I/R in vitro.RESULTSIndicators including fluorescein isothiocyanate-conjugated dextran (4 kilodaltons; FD-4) clearance, ratio of phosphorylated myosin light chain/total myosin light chain, myosin light chain kinase and loss of zonula occludens-1, claudin-2 and VE-cadherin were significantly increased after intestinal I/R or cell hypoxia/reoxygenation. rhANGPTL4 treatment significantly reversed these indicators, which were associated with inhibiting the inflammatory and oxidative cascade, excessive activation of cellular autophagy and apoptosis and improvement of survival rate. Similar results were observed in vitro when cells were challenged by hypoxia/reoxygenation, whereas rhANGPTL4 reversed the indicators close to normal level in Caco-2 cells and human umbilical vein endothelial cells significantly.CONCLUSIONrhANGPTL4 can function as a protective agent against intestinal injury induced by intestinal I/R and improve survival via maintenance of intestinal barrier structure and functions.

Deacidification of Cranberry Juice Reduces Its Antibacterial Properties against Oral Streptococci but Preserves Barrier Function and Attenuates the Inflammatory Response of Oral Epithelial Cells.

Cranberry (Vaccinium macrocarpon) may be a potent natural adjuvant for the prevention of oral diseases due to its anti-adherence, anti-cariogenic, and anti-inflammatory properties. However, the high titrable acidity of cranberry juice (CJ) has been reported to cause gastrointestinal discomfort, leading consumers to restrict their intake of this beverage. Electrodialysis with a bipolar membrane (EDBM) can reduce the organic acid content of CJ while retaining the flavonoids associated with potential health benefits. This study aimed to assess how the deacidification of CJ by EDBM impacts the antibacterial properties of the beverage against cariogenic (Streptococcus mutans, Streptococcus sobrinus) and commensal (Streptococcus gordonii, Streptococcus oralis, Streptococcus salivarius) streptococci, and how it affects oral epithelial barrier function and inflammatory response in an in vitro model. The removal of organic acids from CJ (deacidification rate ≥42%) reduced the bactericidal activity of the beverage against planktonic S. mutans and S. gordonii after a 15-min exposure, whereas only the viability of S. gordonii was significantly impacted by CJ deacidification rate when the bacteria were embedded in a biofilm. Moreover, conditioning saliva-coated hydroxyapatite with undiluted CJ samples significantly lowered the adherence of S. mutans, S. sobrinus, and S. oralis. With respect to epithelial barrier function, exposure to CJ deacidified at a rate of ≥19% maintained the integrity of a keratinocyte monolayer over the course of 24 h compared to raw CJ, as assessed by the determination of transepithelial electrical resistance (TER) and fluorescein isothiocyanate-conjugated dextran paracellular transport. These results can be in part attributed to the inability of the deacidified CJ to disrupt two tight junction proteins, zonula occludens−1 and occludin, following exposure, unlike raw CJ. Deacidification of CJ impacted the secretion of IL-6, but not of IL-8, by oral epithelial cells. In conclusion, deacidification of CJ appears to provide benefits with respect to the maintenance of oral health.

Impact of Processing on the Anti-inflammatory and Gastrointestinal Protective Effects of Cocoa in High Fat-Fed Obese Mice

Abstract Objectives The objective of this study was to determine the impact of fermentation and roasting steps on the anti-inflammatory and gastrointestinal (GI) protective efficacy of dietary cocoa in high fat-fed obese mice. Fermentation and roasting are important for flavor development, but have been shown to reduce total phenolic content in cocoa and are hypothesized to be detrimental for the health beneficial effects of cocoa. To date, however, the available data demonstrating the effect of fermentation and roasting on the anti-inflammatory and GI protective effects of cocoa are limited. Methods Obese male and female C57BL/6J mice were fed a high fat diet (HF) supplemented with one of seven cocoa powders (80 mg/g) that had been processed using different fermentation and roasting parameters. After 9 weeks, GI permeability was determined by gavaging mice with fluorescein isothiocyanate-conjugated dextran and measuring fluorescence in the blood. Markers of colonic inflammation were measured by quantitative reverse-transcriptase PCR. Metabolic endotoxemia was determined by measuring plasma lipopolysaccharide (LPS) and LPS binding protein (LBP). Results Cocoa treatment reduced GI permeability by 48 – 80% in male and female mice compared to HF-fed controls. These differences were paralleled by decreased plasma levels of LPS (25 – 70% lower) and LBP (25 – 57% lower) compared to HF-fed controls. Cocoa-treated mice had reduced colonic mRNA expression of interleukin (IL)1β, IL4, IL6, tumor necrosis factor (TNF)α, and inducible nitric oxide synthase (NOS2) compared HF-fed controls. In contrast to the hypothesis that fermentation and roasting are detrimental to the anti-inflammatory efficacy of cocoa, we found that unfermented/unroasted cocoa and more aggressively fermented/roasted cocoa were equally efficacious at reducing metabolic endotoxemia and colonic expression of IL1β, TNFα, and NOS2. Conclusions In summary, we found that dietary supplementation with cocoa reduces GI permeability, inflammation, and metabolic endotoxemia in HF-fed obese mice, and that the efficacy of cocoa was resilient to fermentation and roasting. Our on-going studies are focused on determining the phytochemical drivers of cocoa bioactivity, and identifying the role of the colonic microbiota as a mediator of the effects of cocoa. Funding Sources The current studies were funded by USDA NIFA.

Partially hydrolyzed guar gum attenuates non-alcoholic fatty liver disease in mice through the gut-liver axis.

BACKGROUNDThe gut-liver axis has attracted much interest in the context of chronic liver disease pathogenesis. Prebiotics such as dietary fibers were shown to attenuate non-alcoholic fatty liver disease (NAFLD) by modulating gut microbiota. Partially hydrolyzed guar gum (PHGG), a water-soluble dietary fiber, has been reported to alleviate the symptoms of various intestinal diseases and metabolic syndromes. However, its effects on NAFLD remain to be fully elucidated.AIMTo determine whether treatment with PHGG attenuates NAFLD development in mice through the gut-liver axis.METHODSSeven-week-old male C57BL/6J mice with increased intestinal permeability were fed a control or atherogenic (Ath) diet (a mouse model of NAFLD) for 8 wk, with or without 5% PHGG. Increased intestinal permeability was induced through chronic intermittent administration of low-dose dextran sulfate sodium. Body weight, liver weight, macroscopic findings in the liver, blood biochemistry [aspartate aminotransferase (AST) and alanine aminotransferase (ALT), total cholesterol, triglyceride, free fatty acids, and glucose levels], liver histology, myeloperoxidase activity in liver tissue, mRNA expression in the liver and intestine, serum endotoxin levels in the portal vein, intestinal permeability, and microbiota and short-chain fatty acid (SCFA) profiles in the cecal samples were investigated.RESULTSMice with increased intestinal permeability subjected to the Ath diet showed significantly increased serum AST and ALT levels, liver fat accumulation, liver inflammatory (tumor necrosis factor-α and monocyte chemotactic protein-1) and fibrogenic (collagen 1a1 and α smooth muscle actin) marker levels, and liver myeloperoxidase activity, which were significantly attenuated by PHGG treatment. Furthermore, the Ath diet combined with increased intestinal permeability resulted in elevated portal endotoxin levels and activated toll-like receptor (TLR) 4 and TLR9 expression, confirming that intestinal permeability was significantly elevated, as observed by evaluating the lumen-to-blood clearance of fluorescein isothiocyanate-conjugated dextran. PHGG treatment did not affect fatty acid metabolism in the liver. However, it decreased lipopolysaccharide signaling through the gut-liver axis. In addition, it significantly increased the abundance of cecal Bacteroides and Clostridium subcluster XIVa. Treatment with PHGG markedly increased the levels of SCFAs, particularly, butyric acid, acetic acid, propionic acid, and formic acid, in the cecal samples.CONCLUSIONPHGG partially prevented NAFLD development in mice through the gut-liver axis by modulating microbiota and downstream SCFA profiles.

Towards the development of a human in vitro model of the blood-brain barrier for virus-associated acute encephalopathy: assessment of the time- and concentration-dependent effects of TNF-α on paracellular tightness.

The pathogenesis of virus-associated acute encephalopathy (VAE) involves brain edema caused by disruption of the blood-brain barrier (BBB). We aimed to develop an in vitro VAE model using an in vitro BBB model, to evaluate the dynamics of vascular dysfunction caused by tumor necrosis factor (TNF)-α. A co-culture model, consisting of Transwell®-grown human brain microvascular endothelial cells and pericytes, was treated with serially diluted TNF-α. Transendothelial electrical resistance (TER) was measured using cellZscope®. A permeability assay, using fluorescein isothiocyanate-conjugated sodium or dextran, was performed. Changes in claudin-5 localization and expression after TNF-α treatment were observed using immunofluorescence staining and western blot analysis. The TER decreased and permeability increased after TNF-α treatment; recovery time was dependent on TNF-α concentration. Claudin-5 was delocalized after TNF-α treatment and recovered in a TNF-α concentration-dependent manner. The expression of claudin-5 decreased 24h after the TNF-α treatment and completely recovered 48h after TNF-α treatment. Claudin-5 delocalization was likely associated with vascular hyperpermeability. To conclude, we evaluated vascular endothelial cell permeability and injury in VAE using an in vitro BBB model treated with TNF-α. This system can be useful for developing novel therapeutic strategies for VAE and designing treatments that target vascular permeability.

ITRAQ-Based Quantitative Proteomic Analysis of Intestines in Murine Polymicrobial Sepsis with Hydrogen Gas Treatment.

ObjectiveSepsis-associated intestinal injury has a higher morbidity and mortality in patients with sepsis, but there is still no effective treatment. Our research team has proven that inhaling 2% hydrogen gas (H2) can effectively improve sepsis and related organ damage, but the specific molecular mechanism of its role is not clear. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics analysis was used for studying the effect of H2 on intestinal injury in sepsis.MethodsMale C57BL/6J mice were used to prepare a sepsis model by cecal ligation and puncture (CLP). The 7-day survival rates of mice were measured. 4-kd fluorescein isothiocyanate-conjugated Dextran (FITC-dextran) blood concentration measurement, combined with hematoxylin-eosinstain (HE) staining and Western blotting, was used to study the effect of H2 on sepsis-related intestinal damage. iTRAQ-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used for studying the proteomics associated with H2 for the treatment of intestinal injury.ResultsH2 can significantly improve the 7-day survival rates of sepsis mice. The load of blood and peritoneal lavage bacteria was increased, and H2 treatment can significantly reduce it. CLP mice had significant intestinal damage, and inhalation of 2% hydrogen could significantly reduce this damage. All 4194 proteins were quantified, of which 199 differentially expressed proteins were associated with the positive effect of H2 on sepsis. Functional enrichment analysis indicated that H2 may reduce intestinal injury in septic mice through the effects of thyroid hormone synthesis and nitrogen metabolism signaling pathway. Western blot showed that H2 was reduced by down-regulating the expressions of deleted in malignant brain tumors 1 protein (DMBT1), insulin receptor substrate 2 (IRS2), N-myc downregulated gene 1 (NDRG1) and serum amyloid A-1 protein (SAA1) intestinal damage in sepsis mice.ConclusionA total of 199 differential proteins were related with H2 in the intestinal protection of sepsis. H2-related differential proteins were notably enriched in the following signaling pathways, including thyroid hormone synthesis signaling pathway, nitrogen metabolism signaling pathways, digestion and absorption signaling pathways (vitamins, proteins and fats). H2 reduced intestinal injury in septic mice by down-regulating the expressions of SAA1, NDRG1, DMBT1 and IRS2.

Altered Properties of Endothelial Cells and Mesenchymal Stem Cells Underlying the Development of Scleroderma-like Vasculopathy in KLF5+/- ;Fli-1+/- Mice.

In prevous studies, we established a new animal model, KLF5+/- ;Fli-1+/- mice, in which fundamental pathologic features of systemic sclerosis (SSc) are broadly recapitulated. SSc vasculopathy is believed to occur as a result of impaired vascular remodeling, but its detailed mechanism of action remains unknown. To address this, the present study investigated the properties of dermal microvascular endothelial cells (DMECs), bone marrow-derived endothelial progenitor cells (BM-EPCs), and bone marrow-derived mesenchymal stem cells (BM-MSCs), a precursor of pericytes, in KLF5+/- ;Fli-1+/- mice. Neovascularization and angiogenesis were assessed in KLF5+/- ;Fli-1+/- mice by in vivo Matrigel plug assay and in vitro tube formation assay, respectively. The properties of mouse BM-EPCs and BM-MSCs were assessed with in vitro studies. Dermal vasculature was visualized in vivo by injecting the mice with fluorescein isothiocyanate-conjugated dextran. Neovascularization was diminished in skin-embedded Matrigel plugs from KLF5+/- ;Fli-1+/- mice. DMECs from KLF5+/- ;Fli-1+/- mice showed defective tubulogenic activity, decreased expression of VE-cadherin and CD31, and an imbalance in the expression of Notch1/Dll4, suggesting that angiogenesis and anastomosis are disturbed. KLF5+/- ;Fli-1+/- mouse BM-MSCs exhibited enhanced proliferation and migration and increased collagen production following stimulation with transforming growth factor β1, indicating that these cells differentiate preferentially into myofibroblasts rather than pericytes. KLF5+/- ;Fli-1+/- mouse BM-EPCs displayed a transition toward mesenchymal cells, suggesting that vasculogenesis is impaired. Wound healing was delayed in KLF5+/- ;Fli-1+/- mice (mean ± SD healing time 15.67 ± 0.82 days versus 13.50 ± 0.84 days; P = 0.0017), and the vascular network was poorly developed in wound scar tissue. The characteristics observed in the KLF5+/- ;Fli-1+/- mouse model - specifically, impaired neovascularization and vascular maturation - are similar to those observed in human SSc, and could be at least partially attributable to the induction of SSc-like properties in DMECs, BM-EPCs, and BM-MSCs. These findings indicate the critical contribution of Klf5 and Fli1 deficiency in vascular cells and related cell precursors to the development of SSc vasculopathy.

Early-life malnutrition causes gastrointestinal dysmotility that is sexually dimorphic.

Slow gastrointestinal (GI) transit occurs in moderate-to-severe malnutrition. Mechanisms underlying malnutrition-associated dysmotility remain unknown, partially due to lack of animal models. This study sought to characterize GI dysmotility in mouse models of malnutrition. Neonatal mice were malnourished by timed maternal separation. Alternatively, low-protein, low-fat diet was administered to dams, with malnourished neonates tested at two weeks or weaned to the same chow and tested as young adults. We determined total GI transit time by carmine red gavage, colonic motility by rectal bead latency, and both gastric emptying and small bowel motility with fluorescein isothiocyanate-conjugated dextran. We assessed histology with light microscopy, ex vivo contractility and permeability with force-transduction and Ussing chamber studies, and gut microbiota composition by 16S rDNA sequencing. Both models of neonatal malnutrition and young adult malnourished males but not females exhibited moderate growth faltering, stunting, and grossly abnormal stomachs. Progression of fluorescent dye was impaired in both neonatal models of malnutrition, whereas gastric emptying was delayed only in maternally separated pups and malnourished young adult females. Malnourished young adult males but not females had atrophic GI mucosa, exaggerated intestinal contractile responses, and increased gut barrier permeability. These sex-specific abnormalities were associated with altered gut microbial communities. Multiple models of early-life malnutrition exhibit delayed upper GI transit. Malnutrition affects young adult males more profoundly than females. These models will facilitate future studies to identify mechanisms underlying malnutrition-induced pathophysiology and sex-specific regulatory effects.

Non-electrophilic TRPA1 agonists, menthol, carvacrol and clotrimazole, open epithelial tight junctions via TRPA1 activation.

Activation of the transient receptor potential A1 channel (TRPA1) by electrophilic agonists was reported to induce the opening of tight junctions (TJs). Because compounds that increase TJ permeability can be paracellular permeability enhancers, we investigated the effect of non-electrophilic TRPA1 activators, including food ingredients (menthol and carvacrol) and medication (clotrimazole), on epithelial permeability. We show that all three compounds induced increase of the permeability of fluorescein isothiocyanate-conjugated dextran (4 kDa) and decrease of transepithelial electrical resistance, accompanied by Ca2+ influx and cofilin activation in epithelial MDCK II monolayers. These phenotypes were attenuated by pretreatment of a TRPA1 antagonist, suggesting TRPA1-mediated opening of TJs. These results suggest that non-electrophilic TRPA1 activators with established safety can be utilized to regulate epithelial barriers.

Osteopontin-loaded PLGA nanoparticles enhance the intestinal mucosal barrier and alleviate inflammation via the NF-κB signaling pathway

Osteopontin is a multifunctional glycoprotein that is secreted by a variety of tissues or cells, but the role of osteopontin in the epithelial mucosal barrier has not been clearly established. We loaded osteopontin into hyaluronic acid-functionalized polymeric nanoparticles, which were administered by gavage to a colitis mouse model. The disease activity index, weight gain and colon length were calculated to assess the degree of symptoms. Epithelial permeability was measured using fluorescein isothiocyanate-conjugated dextran. The enzymatic activity of myeloperoxidase in the colon and inflammatory cytokines were assayed to assess the levels of inflammation. The histological appearance of the colon was observed by H&E staining. Tight junction proteins and signaling pathway proteins (NF-κB and phospho-NF-κB) were determined by western blotting. The resultant spherical osteopontin-loaded nanoparticles were characterized by the expected particle size (approximately 272.3 nm) and a slightly negative zeta potential (approximately -5.3 mV). Interestingly, we found that the osteopontin-loaded nanoparticles exerted remedial effects on colitis by both enhancing the intestinal barrier and alleviating inflammation in vivo according to the tested parameters. These results suggest that OPN plays a positive role in protecting the epithelial mucosal barrier and may be a therapeutic drug in gut homeostasis.

NOX1/NADPH oxidase in bone marrow-derived cells modulates intestinal barrier function

The involvement of reactive oxygen species (ROS) has been suggested in the development of inflammatory bowel disease (IBD). An impaired intestinal barrier function is common in IBD patients. Here, we report the central role of NOX1/NADPH oxidase, a major source of ROS in nonphagocytic cells, in intestinal barrier dysfunction. By in vivo imaging using L-012 as a probe, a time-dependent increase in ROS was demonstrated in the abdomen of wild-type mice (WT) administered lipopolysaccharide (LPS: 6 mg/kg i.p.), but it was almost completely abolished in mice deficient in Nox1 (Nox1-KO) or the inducible nitric oxide synthase gene (iNOS-KO). By ex vivo imaging, increased ROS production was mainly shown in the ileum, where enhanced immunostaining of NOX1 was observed on the apical side of the epithelium. On the other hand, a punctate staining pattern of 3-nitrotyrosine, a marker of peroxynitrite production, was demonstrated in the lamina propria. When LPS-induced intestinal hyperpermeability was assessed by the oral administration of fluorescein isothiocyanate-conjugated dextran (FD-4), it was significantly suppressed in Nox1-KO as well as iNOS-KO. When Nox1-KO adoptively transferred with WT bone marrow were treated with LPS, the serum level of FD-4 was significantly elevated, whereas it remained unchanged in WT receiving bone marrow derived from Nox1-KO. Concomitantly, the activation of matrix metalloproteinase-9 induced by LPS was alleviated not only in intestinal tissue but also in peritoneal macrophages of Nox1-KO. Up-regulation of iNOS by LPS was significantly inhibited in macrophages deficient in Nox1, illustrating a functional hierarchy in NOX1/iNOS signaling. Together, these findings suggest that NOX1 in bone marrow-derived cells, but not epithelial cells, perturbs intestinal barrier integrity during endotoxemia.

Role of α2 receptor in dexmedetomidine-induced improvement in intestinal barrier dysfunction in rats with traumatic brain injury

Objective To evaluate the role of α2 receptor in dexmedetomidine-induced improvement in intestinal barrier dysfunction in rats with traumatic brain injury (TBI). Methods Forty clean-grade healthy Wistar rats, aged 12 weeks, weighing 200-250 g, were divided into 4 groups (n=10 each) using a random number table method: control group (group C), TBI group (group T), dexmedetomidine group (group D) and dexmedetomidine plus atipamezole group (group D+ A). Traumatic brain injury model was established by a 20 g weight free fall impact method in chloral hydrate-anesthetized rats.At 30 min before establishing the model, rats were fed 4.0 kD fluorescein-isothiocyanate-conjugated dextran (FITC-Dex) 60 mg/100 g. Dexmedetomidine 50 μg/kg was injected intraperitoneally at 30 min before establishing the model in D and D+ A groups.Atipamezole 500 μg/kg was injected intraperitoneally at 10 min before injecting dexmedetomidine in group D+ A.Blood samples from common carotid artery were obtained at 6 h after establishing the model for determination of concentrations of epinephrine (E) and norepinephrine (NE) in plasma (by enzyme-linked immunosorbent assay), concentrations of FITC-Dex in serum (using fluorescence spectrophotometry) and activity of diamine oxidase (DAO) in serum (by colorimetry). The small intestine was removed for determination of DAO activity (by colorimetry) and for detection of the expression of tight junctional protein zonula occludens-1 (ZO-1) in the intestinal mucosa of epithelial cells. Results Compared with group C, the concentrations of E and NE in plasma, and FITC-Dex concentration and DAO activity in serum were significantly increased, the DAO activity in intestinal tissues was decreased, and the expression of ZO-1 was down-regulated in T, D and D+ A groups (P<0.05). Compared with group T, the concentrations of E and NE in plasma, and FITC-Dex concentration and DAO activity in serum were significantly decreased, the DAO activity was increased, and the expression of ZO-1 was up-regulated in group D (P<0.05). Compared with group D, the concentrations of E and NE in plasma, and FITC-Dex concentration and DAO activity in serum were significantly increased, the DAO activity was decreased, and the expression of ZO-1 was down-regulated in group D+ A (P<0.05). Conclusion α2 receptor is involved in dexmedetomidine-induced improvement in intestinal barrier dysfunction in rats with TBI. Key words: Receptors, adrenergic, alpha; Dexmedetomidine; Craniocerebral trauma; Intestinal mucosa

Transient receptor potential V4 channel stimulation induces reversible epithelial cell permeability in MDCK cell monolayers.

The transient receptor potential V4 channel (TRPV4) is responsive to a variety of physical and chemical stimuli, including a synthetic agonist GSK1016790A (GSK). Here, we show that TRPV4 is functionally expressed in, and that GSK induces the reversible opening of tight junctions (TJs) in epithelial Madin-Darby canine kidney II monolayers. Stimulation of TRPV4 by GSK induces an increase in fluorescein isothiocyanate-conjugated dextran (4kDa) permeability and a reduction in transepithelial resistance, and these responses are blocked by pretreatment with the specific TRPV4 antagonist. Small conductance, but not large conductance Ca2+ -activated K+ channels, TRPA1 channel, and cofilin activation are involved in TRPV4-mediated reversible opening of TJs. These results suggest that a novel mechanism underlies TRPV4-mediated regulation of the tightness of epithelial barriers.

Cross-Talk between Sirtuin 1 and the Proinflammatory Mediator High-Mobility Group Box-1 in the Regulation of Blood-Retinal Barrier Breakdown in Diabetic Retinopathy

ABSTRACTPurpose: High-mobility group box-1 (HMGB1) mediates inflammation and breakdown of blood-retinal barrier (BRB) in diabetic retina. Sirtuin-1 (SIRT1) has protective effects against inflammation and oxidative stress. The aim of this study was to investigate the interaction between HMGB1 and SIRT1 in regulating BRB breakdown in diabetic retina.Methods: BRB breakdown was assessed in vivo with fluorescein isothiocyanate-conjugated dextran. Vitreous samples from 47 proliferative diabetic retinopathy (PDR) and 19 nondiabetic patients, and epiretinal membranes from 13 patients with PDR were studied by enzyme-linked immunosorbent assay and immunohistochemistry. Retinas from 4-week diabetic rats and from normal rats intravitreally injected with HMGB1 were studied by spectrophotometric assay, Western blot analysis, and RT-PCR. We also studied the effect of the HMGB1 inhibitor glycyrrhizin and the SIRT1 activator resveratrol on diabetes-induced biochemical changes in the retina.Results: HMGB1 levels in vitreous samples from PDR patients were significantly higher than in nondiabetic controls, whereas SIRT1 levels were significantly lower in vitreous samples from patients with inactive PDR than those in patients with active PDR and nondiabetic controls. In epiretinal membranes, SIRT1 was expressed in vascular endothelial cells and stromal cells. Diabetes and intravitreal injection of HMGB1 in normal rats downregulated SIRT1expression, whereas glycyrrhizin and resveratrol normalized diabetes-induced downregulation of SIRT1. Resveratrol significantly attenuated diabetes-induced downregulation of occludin and upregulation of HMGB1 and receptor for advanced glycation end products in the retina and breakdown of BRB.Conclusions: Our findings suggest that a functional link between SIRT1 and HMGB1 is involved in regulating of BRB breakdown in diabetic retina.

TNF-α disrupts the integrity of the porcine respiratory epithelial barrier

The airway epithelium plays an important role in protecting the host against environmental microbial pathogens. The disruption of the epithelial barrier may lead to inflammation and invasion by bacterial pathogens. The aim of the present study was to determine whether TNF-α can damage the integrity of the epithelial barrier using a tracheal epithelial cell model. TNF-α (10 and 100 ng/mL) significantly and time-dependently decreased the transepithelial electrical resistance of the epithelial barrier. The ability of TNF-α to disrupt epithelial barrier integrity was confirmed by quantifying the passage of fluorescein isothiocyanate-conjugated dextran across an epithelial monolayer. Immunofluorescence microscopy showed that TNF-α has a disruptive effect on both occludin and zonula occludens-1, which are important tight junction proteins. In addition to disrupting the epithelial barrier, TNF-α induced the secretion of IL-6 and IL-8 by tracheal epithelial cells. Lastly, incubating Gram-negative respiratory bacterial pathogens with the tracheal epithelial cells caused the cells to secrete TNF-α. In summary, the present study showed that TNF-α, which was secreted by the tracheal epithelial cells when they were stimulated with several swine pathogens, damaged tight junction proteins and disrupted the epithelial barrier of a pig tracheal epithelial cell model. TNF-α can also cause inflammation through its ability to induce IL-6 and IL-8 secretion by epithelial cells.