Monolayer Permeability Research Articles

SMAD3 is a Mediator of TGF‐β ‐induced Barrier Dysfunction and Hyperpermeability in Microvascular Endothelial Cells

The endothelial transcription factor ETS (Erythroblast‐transformation specific)‐related gene (ERG) regulates canonical TGFβ‐SMAD signaling, by repressing SMAD3 activity. SMAD proteins function as important mediators of intracellular signal transduction of TGF‐β. Following their phosphorylation by TGF‐β receptor‐I, Receptor‐regulated SMADs (including SMAD2 and SMAD3 proteins) form a heteromeric complex with co‐SMAD and then translocate into the nucleus where they bind and regulate the expression of multiple target genes. Recent studies from our lab have demonstrated that ERG is a critical regulator of permeability in human lung microvascular endothelial cells (HLMECs) where ERG gene activation provided protection to the barrier. We have hypothesized that the protective effects of ERG on barrier integrity may be mediated via inhibiting TGF‐β/SMAD3 signaling pathway. To study the role of SMAD3 in microvascular endothelial barrier functions and permeability, HLMECs were grown on Transwell inserts and other cell culture platforms. Monolayer permeability was evaluated based on FITC‐albumin extravasation and measurement of its fluorescence intensity. TGF‐β (1‐50 ng/ml) induced a dose‐dependent increase in monolayer hyperpermeability. TGF‐β‐induced hyperpermeability was attenuated by a SMAD3 pharmacological inhibitor (SIS3) or CRISPR/cas9‐mediated knockdown of SMAD3. Immunoblot analysis demonstrated highly efficient SMAD3 knockdown by SMAD3 CRISPR/Cas9. TGF‐β treatment resulted in significant increase in matrixmetalloproteinase‐9 (MMP‐9) activity and the effect was decreased by SMAD3 inhibition. MMP‐9 is known to proteolytically breakdown the adherens junction proteins in microvascular endothelial cells, resulting in hyperpermeability. These results demonstrate that SMAD3 is a mediator of TGF‐β signaling‐induced loss of endothelial barrier integrity and permeability in microvascular endothelial cells. It also suggests that one of the downstream effects of TGFβ‐SMAD signaling is the activation of MMP‐9 which mediates loss of adherens junctional integrity.

Dexmedetomidine reduces dextran sulfate sodium (DSS)-induced NCM460 cell inflammation and barrier damage by inhibiting RhoA/ROCK signaling pathway.

This study investigated the role of dexmedetomidine (DEX) in dextran sulfate sodium (DSS)-induced NCM460 cells. The viability and apoptosis of NCM460 cells treated with DEX with or without DSS were detected by CCK-8 and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The level of inflammatory factors and expression of inflammation-related proteins, tight junction proteins and Ras homolog gene family, member A/Rho-associated coiled-coil containing protein kinase (RhoA/ROCK) signaling-related proteins in NCM460 cells treated with DEX and/or U46619 (RhoA/ROCK agonist) and/or DSS were detected by the respective enzyme-linked immunosorbent assay (ELISA) kits and Western blot analysis. The permeability of NCM460 monolayers was examined with transepithelial electrical resistance (TEER) assay. DEX had no effect on NCM460 cell viability. However, DEX improved the viability and barrier damage and suppressed the apoptosis and inflammation of DSS-induced NCM460 cells. Correspondingly, the expression of inflammation-related proteins was reduced and the expression of tight junction proteins was increased in DSS-induced NCM460 cells after treatment with DEX. In addition, RhoA/ROCK signaling was activated in NCM460 cells induced by DSS, which was suppressed by DEX. The protective effects of DEX on DSS-indued NCM460 cells were reversed by U46619. DEX improved viability and barrier damage while suppressed apoptosis and inflammation in DSS-indued NCM460 cells by inhibiting RhoA/ROCK signaling pathway.

New In Vitro Model to Study Multicellular and Flow Control of Blood‐Brain Barrier

Brain endothelial cells of the blood‐brain barrier (BBB) have been shown to be regulated by supportive cells, such as pericytes and astrocytes, and shear stress exposure. However, studies investigating the impact of pericytes and astrocytes on brain endothelial cell function have identified both beneficial and detrimental results. Additionally, most studies investigating the relationship between shear stress and brain endothelial cell function lack physiological relevance via the use of sub‐physiological shear stress magnitudes and/or via the absence of pericytes and astrocytes. In this study, we developed a millifluidic device compatible with standard transwell inserts to investigate BBB function. In contrast to standard polydimethylsiloxane (PDMS) microfluidic devices, this model allows for easy, reproducible shear stress exposure without common limitations of PDMS devices such as inadequate nutrient diffusion and air bubble formation. In no‐flow conditions, we first used the device to examine the impact of primary human pericytes and astrocytes on human brain microvascular endothelial cell (HBMEC) barrier integrity. We found that astrocytes, pericytes, and a 1:1 ratio of both cell types increased HBMEC barrier integrity via reduced permeability to 40 kDa fluorescent dextran, which was associated with increased expression of tight junction protein, claudin‐5. Interestingly, we also observed a significantly lower permeability to 3 kDa dextran in HBMEC‐pericyte co‐cultures compared to HBMEC‐astrocyte and HBMEC‐astrocyte‐pericyte co‐cultures. Based on these findings, we hypothesize pericytes may be providing increased barrier support to the BBB model compared to astrocytes although they both function as permeability reducers. After using the device to generate 24‐hour flow at 12 dynes/cm2, we observed that shear stress exposure significantly reduced dextran permeability in HBMEC monolayers, but not in tri‐culture models consisting of HBMECs, pericytes, and astrocytes. These results indicate that co‐cultures may demonstrate a more pronounced impact on overall BBB permeability than flow exposure. In both cases, flow exposure was interestingly associated with reduced expression of both claudin‐5 and occludin. However, ZO‐1 expression, and localization at cell‐cell junctions increased in the tri‐culture but exhibited no apparent change in the HBMEC monolayer. Under flow conditions, we also observed alignment of HBMECs in the tri‐culture while no such phenomenon was observed in HBMEC monolayers, indicating supportive cells and flow are both essential to observe brain endothelial cell alignment in vitro. Collectively, these results support the necessity of physiologically relevant, multicellular BBB models when investigating brain endothelial cell function in relation to the BBB. Additionally, our findings provide clues on the role of shear stress and supportive cells within the BBB, a critical step to elucidating the physiology of the neurovascular unit.

Andrographolide‐Mediated Protection of Barrier Integrity and Permeability is Mediated Through NLRP3 Inflammasome Inhibition in Blood‐Brain Barrier Endothelial Cells

The blood‐brain barrier (BBB) that consists of the inter‐endothelial tight junctions, acts as the major blood‐brain interface and regulator of brain microvascular hyperpermeability. In the brain, the loss of BBB integrity and hyperpermeability leads to cerebral edema formation and intracranial pressure elevation in traumatic brain injury and ischemic strokes. Recent studies demonstrated that reactive oxygen species are potential signals for the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation that leads to interleukin‐1β (IL‐1β) secretion. IL‐1β plays a major role in promoting BBB hyperpermeability in various brain pathologies, including TBI. Furthermore, recent studies from our lab have demonstrated a tri‐phasic role for hydrogen peroxide (H2O2) in BBB endothelial cells where it promotes, angiogenesis, hyperpermeability and apoptosis in a tri‐phasic and concentration dependent manner. Hydrogen peroxide plays a major role physiologically as the second messenger and pathologically as an inducer of oxidative stress and associated cellular signaling. We hypothesized that in BBB endothelial cells, H2O2‐induced barrier dysfunction and hyperpermeability are NLRP3 inflammasome‐dependent. The main objective of our study was to evaluate the role of NLRP3 inflammasome signaling in H2O2‐mediated barrier dysfunction and hyperpermeability in human brain microvascular endothelial cells and to test whether andrographolide, a labdane diterpenoid possessing potent anti‐inflammatory properties will attenuate such effects. Th effect of H2O2 treatment on tight junction integrity/permeability was studied using zonula occludens‐1 (ZO‐1) immunofluorescence localization, ZO‐1 immunoblot analysis, Transwell monolayer permeability assay using FITC‐dextran (10‐kDa) as a fluorescent marker and cell viability/apoptosis assay. The effect of NLRP3 inhibitor (MCC950) and andrographolide on H2O2‐induced hyperpermeability was studied using Transwell permeability assay. Our results show that, CRISPR/Cas‐9‐mediated knockdown of ZO‐1 resulted in monolayer hyperpermeability demonstrating the significance of ZO‐1 in regulating barrier functions. CRISPR‐based activation of NLRP3 induced monolayer hyperpermeability whereas CRISPR/Cas‐9‐based NLRP3 knockdown had no significant effect on permeability. H2O2 (10µM) induced monolayer hyperpermeability and the effect was decreased by MCC950 and andrographolide treatment. Hydrogen peroxide induced cathepsin B (an activator of the NLRP3 pathway) activity significantly, and the effect was decreased by andrographolide treatment. H2O2‐mediated barrier dysfunction and hyperpermeability were not due to changes in cell viability/apoptosis. These results suggest that NLRP3 is a mediator of H2O2‐induced barrier dysfunction and hyperpermeability in human BBB endothelial cells and andrographolide is an effective inhibitor of this pathway.

The development of an intestinal microphysiological system to model oral drug disposition

The intestine has important gate‐keeping functions that can profoundly impact the systemic blood exposure of orally administered drugs. Over the past decade, there has been growing effort to develop advanced in vitro tissue models that better predict in vivo DMET processes. One such novel model is the microphysiological system (MPS), which provides a more physiologically and anatomically relevant epithelial cell culture platform than traditional, static cell culture models. The MPS incorporates flow of media down luminal and abluminal channels, with accompanying shear forces that have been shown to be beneficial for multicellular viability and function. In this study, we cultured the immortalized cell line, LS180, in the Microfluidic ChipShop’s “Fluidic 480” MPS. LS180 cells have demonstrated high metabolic activity, particularly for CYP3A substrates, but display poor performance for monolayer permeability studies due to a lack of tight junction formation and uncertain transporter function. We assessed CYP3A metabolic activity, cell monolayer integrity, and global RNA expression for LS180 cells cultured in the MPS versus traditional TranswellTM culture. To measure CYP3A activity, we incubated LS180 cells cultured MPS and TranswellTM with the CYP3A probe substrate, midazolam, and monitored total product formation and parent midazolam appearance in the basolateral media of the two culturing platforms (with static or dynamic flow). LS180 cells cultured in MPS display a 2‐3‐fold greater midazolam extraction ratio (ER) compared to LS180 cells cultured in TranswellTM. We then compared cell monolayer integrity in the two culture platforms by measuring the apparent permeability (Papp) of the paracellular transport probe, atenolol. LS180 cells cultured in MPS display a 3‐fold lower Papp for atenolol than LS180 cells cultured in TranswellTM, indicating improved barrier integrity and tight junction formation for LS180 cells cultured in MPS. Using RNA‐seq, we compared global RNA expression for LS180 cells cultured in MPS versus TranswellTM. There were minimal expression differences for phase I and II metabolic enzymes between the two groups. Interestingly, transporter expression differed greatly between the two groups, with some transporters displaying greater expression in MPS compared to TranswellTM (e.g., P‐gp, MRP‐2) while others displayed a decrease in expression (e.g., OATP2B1, ENT1). Further testing is required to assess whether the changes in transporter RNA expression leads to changes in transporter protein abundance and localization. The sum of these results suggest that culturing LS180 cells in a MPS improves their performance and ability to predict the disposition of orally administered drugs.

Preclinical In Vitro Model to Assess the Changes in Permeability and Cytotoxicity of Polarized Intestinal Epithelial Cells during Exposure Mimicking Oral or Intravenous Routes: An Example of Arsenite Exposure.

The gastrointestinal tract (GIT) is exposed to xenobiotics, including drugs, through both: local (oral) and systemic routes. Despite the advances in drug discovery and in vitro pre-clinical models, there is a lack of appropriate translational models to distinguish the impact of these routes of exposure. Changes in intestinal permeability has been observed in different gastrointestinal and systemic diseases. This study utilized one such xenobiotic, arsenic, to which more than 200 million people around the globe are exposed via their food, drinking water, work environment, soil, and air. The purpose of this study was to establish an in vitro model to mimic gastrointestinal tract exposure to xenobiotics via oral or intravenous routes. To achieve this, we compared the route (mimicking oral and intravenous exposure to GIT and the dose response (using threshold approach) of trivalent and pentavalent inorganic arsenic species on the permeability of in vitro cultured polarized T84 cells, an example of intestinal epithelial cells. Arsenic treatment to polarized T84 cells via the apical and basolateral compartment of the trans-well system reflected oral or intravenous routes of exposure in vivo, respectively. Sodium arsenite, sodium arsenate, dimethyl arsenic acid sodium salt (DMAV), and disodium methyl arsonate hydrate (MMAV) were assessed for their effects on intestinal permeability by measuring the change in trans-epithelial electrical resistance (TEER) of T-84 cells. Polarized T-84 cells exposed to 12.8 µM of sodium arsenite from the basolateral side showed a marked reduction in TEER. Cytotoxicity of sodium arsenite, as measured by release of lactate dehydrogenase (LDH), was increased when cells were exposed via the basolateral side. The mRNA expression of genes related to cell junctions in T-84 cells was analyzed after exposure with sodium arsenite for 72 h. Changes in TEER correlated with mRNA expression of focal-adhesion-, tight-junction- and gap-junction-related genes (upregulation of Jam2, Itgb3 and Notch4 genes and downregulation of Cldn2, Cldn3, Gjb1, and Gjb2). Overall, exposure to sodium arsenite from the basolateral side was found to have a differential effect on monolayer permeability and on cell-junction-related genes as compared to apical exposure. Most importantly, this study established a preclinical human-relevant in vitro translational model to assess the changes in permeability and cytotoxicity during exposure, mimicking oral or intravenous routes.

Midazolam Ameliorates Impairment of the Blood-Brain Barrier (BBB) Against LPS.

Central nervous system (CNS) dysfunction induced by sepsis and pathogenic microbial infections is reported to be closely associated with increased permeability of the blood-brain barrier (BBB), which is mainly mediated by the stimulation of lipopolysaccharide (LPS) on inflammatory signaling. Midazolam is a novel sedative acting on the benzodiazepine receptor, which is recently reported to exert a neuroprotective effect by inhibiting inflammation. The present study will explore the potential repair capacity of Midazolam on LPS-induced damage to the BBB. The in vivo mice model was established by intraperitoneal injection of LPS, while the in vitro model was constructed by stimulating endothelial cells utilizing LPS. We found that the increased malondialdehyde (MDA) level and reduced superoxide dismutase (SOD) activity in the brain cortices, promoted serum concentration of inflammatory factors, and elevated BBB permeability were found in the LPS group, all of which were dramatically reversed by 1mg/kg and 2mg/kg Midazolam. Interestingly, Midazolam increased the expression of the tight junction protein zonula occludens-1 (ZO-1). In LPS-challenged in vitro human brain microvascular endothelial cells (HBMECs), the increased concentration of inflammatory factors, reduced trans-endothelial electrical resistance (TEER) level, elevated relative value of trans-endothelial permeability, and downregulated ZO-1 were observed, all of which were pronouncedly alleviated by Midazolam, accompanied by the inhibition on the Ras homolog family member A/ Rho-kinase 2 (RhoA/ROCK-2) pathway. Furthermore, the regulatory effects of Midazolam on ZO-1 expression and the endothelial monolayer permeability in LPS-challenged HBMECs were abolished by the overexpression of RhoA. Collectively, our data imply that Midazolam ameliorated the impairment of the BBB against LPS by regulating the RhoA/ROCK2 pathway.

Effects of Lipolysis-Stimulated Lipoprotein Receptor on Tight Junctions of Pancreatic Ductal Epithelial Cells in Hypertriglyceridemic Acute Pancreatitis.

Objective We investigated the effects of lipolysis-stimulated lipoprotein receptor (LSR) on the tight junctions (TJs) of pancreatic ductal epithelial cells (PDECs) in hypertriglyceridemic acute pancreatitis (HTGAP). Methods Sprague-Dawley rats were fed standard rat chow or a high-fat diet and injected with sodium taurocholate to obtain normal and HTGAP rats, respectively. Serum triglyceride (TG) levels, pathological changes, TJ proteins in the pancreas, and TJ ultrastructure of PDECs were assessed. LSR overexpression (OE) and knockdown (KD) HPDE6-C7 models were designed and cultured in a high-fat environment. Protein levels were quantified by Western blotting. Cell monolayer permeability was detected using FITC-Dextran. Results Serum TG concentration and pancreatic scores were higher in the HTGAP group than in the normal group. Among the TJ proteins, LSR protein expression was significantly lower in the HTGAP group than in the acute pancreatitis (AP) group. Tricellulin (TRIC) expression in the pancreatic ductal epithelia was higher in the HTGAP group than in the AP group. The HTGAP group had lower TJ protein levels, wider intercellular space, and widespread cellular necrosis with disappearance of cell junction structures. In the cell study, TJ proteins were downregulated and the cellular barrier was impaired by palmitic acid (PA), which was reversed by LSR-OE, whereas LSR-KD downregulated the TJ proteins and aggravated PA-induced cellular barrier impairment. Conclusions Hypertriglyceridemia downregulates the TJ proteins in PDECs, which may impair the pancreatic ductal mucosal barrier function. LSR regulation can change the effects of HTG on cellular barrier function by upregulating the TJ proteins.

Galactooligosaccharide Treatment Alleviates DSS-Induced Colonic Inflammation in Caco-2 Cell Model

The biological activities of dietary bioactive polysaccharides have been largely explored. Studies on the immunomodulating effects of oligosaccharides and polysaccharides have shown that they are able to modulate innate immunity. Prebiotics are a class of poorly digested carbohydrates that are mainly produced from dietary fibers, which are carbohydrate polymers with ten or more monomeric units as defined by the Codex Alimentarius Commission in 2009. Considering the capacity of prebiotics in reducing gut inflammation, the aim of this study was to investigate the anti-inflammatory activity of galactooligosaccharide (Bimuno® GOS) in an in vitro model of ulcerative colitis (UC)-like inflamed intestinal cells. Differentiated Caco-2 cells were exposed to 2 % dextran-sulfate-sodium salt (DSS) to induce inflammation, and then with different concentrations of Bimuno GOS (1–1,000 μg/ml). Cell monolayer permeability, tight- and adherent junction protein distribution, pro-inflammatory cytokine secretion, and NF-kB cascade were assessed. Bimuno GOS at different concentrations, while not affecting cell monolayer permeability, was shown to counteract UC-like intestinal inflammatory responses and damages induced by DSS. Indeed, Bimuno GOS was able to counteract the detrimental effects of DSS on cell permeability, determined by transepithelial electrical resistance, phenol red apparent permeability, and tight- and adherent junction protein distribution. Furthermore, Bimuno GOS inhibited the DSS-induced NF-kB nuclear translocation and pro-inflammatory cytokine secretion. Further analyses showed that Bimuno GOS was able to revert the expression levels of most of the proteins involved in the NF-kB cascade to control levels. Thus, the prebiotic Bimuno GOS can be a safe and effective way to modulate the gut inflammatory state through NF-kB pathway modulation, and could possibly further improve efficacy in inducing remission of UC.

44 Endothelial Monolayers Treated with Burn Patient Plasma Exhibit Differential Gene Expression Linked to Cytoskeletal Rearrangement

IntroductionBurn shock is one of the most serious and complex complications suffered by patients following thermal injury. Endothelial dysfunction may play a role in the pathogenesis of burn shock. However, the mechanisms underlying the contribution to pathophysiology are still largely unknown. Previous studies have shown a connection between the rearrangement of cytoskeletal elements leading to increased vascular permeability. The aim of this study was to examine the differential expression of genes involved in cytoskeletal arrangement in endothelial cell monolayers treated with plasma from burn patients.MethodsHuman umbilical vein endothelial cells (HUVECs) were seeded into transwell plates to form confluent monolayers. Plasma was collected from burn patients 4 hours post-admission. HUVEC cells were exposed to 10% multi-donor pooled healthy human plasma (HHP) or burn patient plasma. Monolayers were subsequently incubated with FIT-C Dextran (40,000 kD) for 2 hours. Monolayer permeability was measured with indices calculated by normalizing values to blank wells (transwell inserts) and HHP-treated monolayer FIT-C diffusion. RNA was isolated from these same cells that had increased monolayer permeability and PCR analysis was carried out using an 84 gene array of human cytoskeletal regulators (Qiagen). A Ct value of 35 was used to indicate expression and a fold change of 1.5 to indicate differential expression in the control vs. injured groups.ResultsFour burn patient plasma samples were utilized to create injuries. Patients were mostly male (75%) with a mean age of 50±20 years and mean %TBSA burn of 37±34%. Differential gene expression in burn vs. HHP was compared. Ten genes showed significant upregulation (ARHGDIB, AURKA, AURKB, CCNB2, CIT, IQGAP2, VASP, SSH2, MYLK and DIAPH1). Four genes showed significant downregulation in (FSCN2, ARHGAP6, CYFIP2, CCNA1). Monolayer permeability indices showed statistically significant increases when compared to controls ranging from 3-13.33% (p < .05).ConclusionsThe interplay of burn shock and endothelial dysfunction remains a complex process of which much is unknown. However, RNA analyses of burn patient plasma reveals involvement of multiple cytoskeletal regulators. Furthermore, all these samples show a concurrent increase in permeability indices when compared to controls, further strengthening the association between cytoskeletal rearrangement and endotheliopathy. Future research to better understand the specifics of these pathways could help aid in the development of more targeted treatments of endotheliopathy and burn shock. Cytoskeletal rearrangement may be an interesting target for future work to understand this mechanistic interplay.

Effects of exosomes from human adipose-derived mesenchymal stem cells on pulmonary vascular endothelial cells injury in septic mice and its mechanism

Effects of exosomes from human adipose-derived mesenchymal stem cells on pulmonary vascular endothelial cells injury in septic mice and its mechanism

Comparison of the Drug-Induced Efficacies between Omidenepag Isopropyl, an EP2 Agonist and PGF2α toward TGF-β2-Modulated Human Trabecular Meshwork (HTM) Cells.

To compare the drug-induced efficacies between omidenepag (OMD), an EP2 agonist, and prostaglandin F2α (PGF2α) on glaucomatous trabecular meshwork (TM) cells, two- and three-dimensional (2D and 3D) cultures of TGF-β2-modulated human trabecular meshwork (HTM) cells were used. The following analyses were performed: (1) transendothelial electrical resistance (TEER) and FITC-dextran permeability measurements (2D), (2) the size and stiffness of the 3D spheroids, and (3) the expression (both 2D and 3D) by several extracellular matrix (ECM) molecules including collagen (COL) 1, 4 and 6, and fibronectin (FN), and α smooth muscle actin (αSMA), tight junction (TJ)-related molecules, claudin11 (Cldn11) and ZO1, the tissue inhibitor of metalloproteinase (TIMP) 1–4, matrix metalloproteinase (MMP) 2, 9 and 14, connective tissue growth factor (CTGF), and several endoplasmic reticulum (ER) stress-related factors. TGF-β2 significantly increased the TEER values and decreased FITC-dextran permeability, respectively, in the 2D HTM monolayers, and induced the formation of downsized and stiffer 3D HTM spheroids. TGF-β2-induced changes in TEER levels and FITC-dextran permeability were remarkably inhibited by PGF2α. PGF2α induced increases in the sizes and stiffness of the TGF-β2-treated 3D spheroids, but OMD enhanced only spheroid size. Upon exposure to TGF-β2, the expression of most of the molecules that were evaluated were significantly up-regulated, except some of ER stress-related factors were down-regulated. TJ-related molecules or ER stress-related factors were significantly up-regulated (2D) or down-regulated (3D), and down-regulated (2D) by PGF2α and OMD, while both drugs altered the expression of some of the other genes in the 3D spheroids in a different manner. The findings presented herein suggest that PGF2α and OMD differently modulate the permeability of the TGFβ2-modulated 2D monolayers and the physical properties of the 3D HTM spheroids.

Periductal bile acid exposure causes cholangiocyte injury and fibrosis.

Bile duct integrity is essential for the maintenance of the structure and function of the biliary tree. We previously showed that cholangiocyte injury in a toxic model of biliary atresia leads to increased monolayer permeability. Increased epithelial permeability was also shown in other cholangiopathies. We hypothesized that after initial cholangiocyte injury, leakage of bile acids into the duct submucosa propagates cholangiocyte damage and fibrosis. We thus aimed to determine the impact of bile acid exposure on cholangiocytes and the potential therapeutic effect of a non-toxic bile acid. Extrahepatic bile duct explants were isolated from adult and neonatal BALB/c mice. Explants were cultured with or without glycochenodeoxycholic acid and ursodeoxycholic acid. They were then fixed and stained. Explants treated with glycochenodeoxycholic acid demonstrated cholangiocyte injury with monolayer disruption and partial lumen obstruction compared to control ducts. Masson's trichrome stains revealed increased collagen fibers. Myofibroblast marker α-SMA stains were significantly elevated in the periductal region. The addition of ursodeoxycholic acid resulted in decreased cholangiocyte injury and reduced fibrosis. Bile acid leakage into the submucosa after initial cholangiocyte injury may serve as a possible mechanism of disease propagation and progressive fibrosis in cholangiopathies.

Curcumin Mitigates TNFα-Induced Caco-2 Cell Monolayer Permeabilization Through Modulation of NF-κB, ERK1/2, and JNK Pathways.

This work studies the capacity of curcumin to inhibit tumor necrosis alpha (TNFα)-induced inflammation, oxidative stress, and loss of intestinal barrier integrity, characterizing the underlying mechanisms. Caco-2 cell monolayers are incubated with TNFα (10ng mL-1 ), in the absence or presence of curcumin. TNFα causes an increase in interleukin (IL)-6 and IL-8 release, which is inhibited by curcumin in a dose-dependent manner (half-maximal inhibitory concentration (IC50 ) = 3.4 µM for IL-6). Moreover, TNFα leads to: i) increased intercellular adhesion molecule 1 (ICAM-1) and NLRP3 inflammasome expression; ii) increased cell monolayer permeability and decreased levels of tight junction proteins; iii) increased cellular and mitochondrial oxidant production; iv) decreased mitochondrial membrane potential and complex I-III activity; v) activation of redox-sensitive pathways, i.e., nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun N-terminal kinases (JNK); and vi) increased myosin light-chain kinase (MLCK) expression and phosphorylation levels of myosin light-chain protein MLC. Curcumin (2-8 µM) inhibits all these TNFα-triggered undesirable outcomes, mostly showing dose-dependent effects. The inhibition of NF-κB, ERK1/2, and JNK activation could be in part involved in the capacity of curcumin to mitigate intestinal inflammation, oxidant production, activation of redox-sensitive pathways, and prevention of monolayer permeabilization. These results support an action of dietary curcumin in sustaining gastrointestinal tract physiology.

Endothelial glycocalyx is damaged in diabetic cardiomyopathy: angiopoietin 1 restores glycocalyx and improves diastolic function in mice

Aims/hypothesisDiabetic cardiomyopathy (DCM) is a serious and under-recognised complication of diabetes. The first sign is diastolic dysfunction, which progresses to heart failure. The pathophysiology of DCM is incompletely understood but microcirculatory changes are important. Endothelial glycocalyx (eGlx) plays multiple vital roles in the microcirculation, including in the regulation of vascular permeability, and is compromised in diabetes but has not previously been studied in the coronary microcirculation in diabetes. We hypothesised that eGlx damage in the coronary microcirculation contributes to increased microvascular permeability and hence to cardiac dysfunction.MethodsWe investigated eGlx damage and cardiomyopathy in mouse models of type 1 (streptozotocin-induced) and type 2 (db/db) diabetes. Cardiac dysfunction was determined by echocardiography. We obtained eGlx depth and coverage by transmission electron microscopy (TEM) on mouse hearts perfusion-fixed with glutaraldehyde and Alcian Blue. Perivascular oedema was assessed from TEM images by measuring the perivascular space area. Lectin-based fluorescence was developed to study eGlx in paraformaldehyde-fixed mouse and human tissues. The eGlx of human conditionally immortalised coronary microvascular endothelial cells (CMVECs) in culture was removed with eGlx-degrading enzymes before measurement of protein passage across the cell monolayer. The mechanism of eGlx damage in the diabetic heart was investigated by quantitative reverse transcription-PCR array and matrix metalloproteinase (MMP) activity assay. To directly demonstrate that eGlx damage disturbs cardiac function, isolated rat hearts were treated with enzymes in a Langendorff preparation. Angiopoietin 1 (Ang1) is known to restore eGlx and so was used to investigate whether eGlx restoration reverses diastolic dysfunction in mice with type 1 diabetes.ResultsIn a mouse model of type 1 diabetes, diastolic dysfunction (confirmed by echocardiography) was associated with loss of eGlx from CMVECs and the development of perivascular oedema, suggesting increased microvascular permeability. We confirmed in vitro that eGlx removal increases CMVEC monolayer permeability. We identified increased MMP activity as a potential mechanism of eGlx damage and we observed loss of syndecan 4 consistent with MMP activity. In a mouse model of type 2 diabetes we found a similar loss of eGlx preceding the development of diastolic dysfunction. We used isolated rat hearts to demonstrate that eGlx damage (induced by enzymes) is sufficient to disturb cardiac function. Ang1 restored eGlx and this was associated with reduced perivascular oedema and amelioration of the diastolic dysfunction seen in mice with type 1 diabetes.Conclusions/interpretationThe association of CMVEC glycocalyx damage with diastolic dysfunction in two diabetes models suggests that it may play a pathophysiological role and the enzyme studies confirm that eGlx damage is sufficient to impair cardiac function. Ang1 rapidly restores the CMVEC glycocalyx and improves diastolic function. Our work identifies CMVEC glycocalyx damage as a potential contributor to the development of DCM and therefore as a therapeutic target.Graphical abstract

Improvement of the pharmacokinetics and antifibrotic effects of nintedanib by intrapulmonary administration of a nintedanib–hydroxypropyl-γ-cyclodextrin inclusion complex in mice with bleomycin-induced pulmonary fibrosis

Idiopathic pulmonary fibrosis is a chronic lung disease that is characterized by progressive abnormal reprogramming following injury of the pulmonary structure. In this study, we prepared a nintedanib (antifibrotic agent) and cyclodextrin (CyD) inclusion complex to improve the pharmacokinetics and antifibrotic effects of nintedanib following intrapulmonary administration. Hydroxypropyl-γ-CyD (HP-γ-CyD) enhanced the solubility of nintedanib without cytotoxic effects on WI-38 cells (lung fibroblasts) and NCI-H441 cells (alveolar epithelium model). Compared with nintedanib ethanesulfonate salt, the nintedanib–HP-γ-CyD inclusion complex exhibited prolonged distribution in the lungs following intrapulmonary administration in mice with bleomycin-induced pulmonary fibrosis. In addition, compared with nintedanib ethanesulfonate salt, the nintedanib–HP-γ-CyD inclusion complex exhibited higher stability in the bronchoalveolar lavage fluid and lower permeability in NCI-H441 cell monolayers. These results suggested that the inclusion complexation of nintedanib into HP-γ-CyD improved its pharmacokinetics following intrapulmonary administration by increasing its stability in the lungs and reducing its permeability through the alveolar cell membrane. Intrapulmonary administration of the nintedanib–HP-γ-CyD inclusion complex significantly reduced the intrapulmonary hydroxyproline content and limited pathological fibrotic changes. Overall, this study indicates that antifibrotic agent–CyD inclusion complexation intended for intrapulmonary administration can be used to prolong distribution in the lungs and lead to the expansion of idiopathic pulmonary fibrosis therapy.

In Vitro Methods for Measuring the Permeability of Cell Monolayers.

Cell monolayers, including endothelial and epithelial cells, play crucial roles in regulating the transport of biomolecules to underlying tissues and structures via intercellular junctions. Moreover, the monolayers form a semipermeable barrier across which leukocyte transmigration is tightly regulated. The inflammatory cytokines can disrupt the epithelial and endothelial permeability, thus the reduced barrier integrity is a hallmark of epithelial and endothelial dysfunction related with numerous pathological conditions, including cancer-related inflammation. Therefore, the assessment of barrier function is critical in in vitro models of barrier-forming tissues. This review summarizes the commercially available in vitro systems used to measure the permeability of cellular monolayers. The presented techniques are separated in two large groups: macromolecular tracer flux assays, and electrical impedance measurement-based permeability assays. The presented techniques are briefly described and compared.

Enhanced water dispersibility and Caco-2 cell monolayer permeability of quercetin by complexation with casein hydrolysate.

Although quercetin (Que) has many beneficial therapeutic effects on the human body, its oral bioavailability is poor because of its low water solubility. Herein, we describe the preparation of casein hydrolysate (Pepcas ) and its use as a dispersant to enhance the water dispersibility of Que resulting in an improvement of its bioavailability. By complexation with Pepcas , the apparent solubility of Que in the complex (Que@Pepcas ) was significantly increased under acidic and neutral conditions. Que@Pepcas was pre-digested in vitro using a simulated digestive juice, and a Caco-2 cell monolayer permeation assay was performed to evaluate the oral bioavailability of Que. The cumulative amount of Que that permeated the monolayer using Que@Pepcas after 8, 16, and 24 h was increased compared with a blank sample (Que@Blank), and the apparent permeation coefficient Papp of Que@Pepcas was approximately 2.6-fold better than that of Que@Blank. PRACTICAL APPLICATION: Pepcas can be used as a high absorption supplement and food additive, and our Pepcas complexation technique should be widely applicable to not only Que but also to other poorly water-soluble substances.

Extracellular histones cause intestinal epithelium injury and disrupt its barrier function in vitro and in vivo

Extracellular histones are cytotoxic to various cells and have been extensively proven a vital mediator of multiple organ injuries. However, the effect of extracellular histones on the intestine remains largely unknown. This study aimed to clarify the effect of extracellular histones on the intestine. IEC-6, a cell line of rat small intestinal epithelial crypt, and C57BL/6 or ICR mice were treated with histones. The IEC-6 cells treated with histones from 20 μg/mL to 200 μg/mL for 0−24 h displayed a decline of cell viability and an increase of cell death in a concentration- and time-dependent manner. Moreover, histones (100 μg/mL) induced IEC-6 apoptosis through activating caspase 3 and necroptosis through up-regulation of receptor-interacting serine/threonine protein kinase 1 and 3 (RIPK1 and RIPK3), phosphorylated mixed-lineage kinase domain-like protein (p-MLKL) along with the decrease of caspase-8. Histones treatment disturbed zonular occludens 1 (ZO-1) expression and increased permeability of IEC-6 cell monolayer. In vivo, histones 50 mg/kg injection caused mice intestinal edema, loss apex of villus, epithelial lifting down the sides of the villi, and increased neutrophil infiltration. Elevation of serum intestinal fatty acid binding protein (I-FABP), d-lactate, or Diamine oxidase (DAO) and loss of tight junction protein, ZO-1, at 3 h and 6 h after histones injection strongly indicated severe intestinal epithelium injury, which led to increased permeability of the intestine. In conclusion, extracellular histones cause intestinal epithelial damage via direct cytotoxicity. Consequently, intestinal epithelial tight junction and barrier integrity are disrupted, which may play pivotal roles in diverse diseases.

The protective effects of Mirtazapine against lipopolysaccharide (LPS)-induced brain vascular hyperpermeability

ABSTRACT Sepsis is mainly characterized by severe inflammation triggered by infection, and sepsis-associated encephalopathy (SAE) is defined as brain damage caused by sepsis. Disruption of the blood-brain barrier (BBB) triggered by injured brain microvascular endothelial cells (BMECs) and damaged tight junction (TJ) structure is closely associated with the pathogenesis of SAE. The present research proposed to evaluate the potential therapeutic effects of Mirtazapine, a central presynaptic α2 receptor antagonist, on LPS-induced BBB disruption. The mice were administered with normal saline and 10 mg/kg Mirtazapine for 8 consecutive days, and from day 6, the experiment group of mice received LPS for 2 days to induce SAE. We found that the increased BBB permeability, elevated concentrations of inflammatory factors in brain tissues, and downregulated zonula occludens −1 (ZO-1) were observed in LPS-stimulated mice, all of which were reversed by 10 mg/kg Mirtazapine. In the in vitro assay, bEnd.3 brain endothelial cells were treated with 1 μM LPS in the absence or presence of Mirtazapine (25, 50 μM). We found that LPS-treated cells had significantly declined transendothelial electrical resistance (TEER), increased monolayer permeability, elevated production of inflammatory factors, and downregulated ZO-1. However, 25 and 50 μM Mirtazapine ameliorated all these LPS- induced aberrations. Mirtazapine also mitigated the decreased level of NF-E2–related factor 2 (Nrf2) in LPS-challenged endothelial cells. The protective effect of Mirtazapine on endothelial permeability against LPS was significantly abolished by the knockdown of Nrf2. Collectively, we concluded that Mirtazapine exerted protective effects on LPS-induced endothelial cells hyperpermeability by upregulating Nrf2.